Antibodies to 25-hydroxyvitamin D2 and D3 and uses thereof

ABSTRACT

Provided herein are antigenic molecules that can be used to generate antibodies capable of binding to a vitamin D derivative, such as 25-hydroxyvitamin D2 and/or 25-hydroxyvitamin D3, or a 25-hydroxyvitamin D analog, such as a vitamin D-C22 immunogenic molecule or compound. Antibodies produced using these antigenic molecules, and related antigenic compounds, are also described. In addition, disclosed herein are methods for detecting vitamin D deficiency in a subject, methods for treating a subject suspected of having a vitamin D deficiency, methods for monitoring progression of vitamin D deficiency in a subject, and methods for monitoring treatment of vitamin D deficiency in a subject in need thereof. Also provided are methods and reagents for the detection or quantification of 25-hydroxyvitamin D2 and D3, methods for stabilizing vitamin D analogs, and methods for separating 25-hydroxyvitamin D2 and D3 from vitamin D binding protein in a biological sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/488,630 filed May 20, 2011, the content of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

Provided herein are methods and reagents for the detection orquantification of 25-hydroxyvitamin D2 and D3, methods for stabilizingvitamin D analogs, and methods for separating 25-hydroxyvitamin D2 andD3 from vitamin D binding protein in a biological sample.

BACKGROUND

Vitamin D is a steroid hormone involved in intestinal absorption ofcalcium and regulation of calcium homeostasis. Vitamin D is essentialfor the formation and maintenance of strong, healthy bones.

Vitamin D deficiency can result from inadequate exposure to the sun,inadequate alimentary intake, decreased absorption, abnormal metabolism,or vitamin D resistance. Vitamin D deficiency has been linked torickets, osteomalacia, osteoporosis, high blood pressure, cardiovasculardisease, schizophrenia, depression, nervous system diseases, diabetes,infectious diseases, asthma, allergies, cancer, and several autoimmunediseases.

Whether consumed or produced, both forms of vitamin D (D2 and D3) aremetabolized by the liver to 25-hydroxyvitamin D (25(OH)D) and thenconverted in the liver or kidney to 1,25-dihydroxyvitamin D. Vitamin Dmetabolites are bound to a carrier protein in the plasma and distributedthroughout the body. It is generally accepted that 25(OH)D is themetabolite that is the most reliable clinical indicator of vitamin Dstatus because the serum 25(OH)D levels reflect the body's storagelevels of vitamin D and correlate with clinical symptoms of vitamin Ddeficiency.

Despite the value of detection of vitamin D to health management,accurate and sensitive assays for the detection of vitamin D or itsderivatives are limited. One obstacle to development of successfulassays for vitamin D has been the technical difficulty in the isolationof tightly bound 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 fromtheir vitamin D binding protein (DBP) in test biological samples. DBP isa serum glycoprotein that binds vitamin D sterols, G-actin, fatty acidsand chemotactic agents. Swamy et al., Archives of Biochemistry andBiophysics 402: 14-23 (2002). In plasma, 25-hydroxyvitamin D3 and25-hydroxyvitamin D2 have a half-life of two to three weeks, and yet areonly present in less than 0.05% free form. The majority is bound to DBPwith an association affinity as high as 10⁹ M⁻¹ which involves hydrogenbinding as well as hydrophobic interactions.

Another obstacle in developing a competitive binding immunoassay forvitamin D is the instability of the vitamin D analog used to compete forantibody binding sites with the vitamin D in biological samples. VitaminD in the absence of DBP is highly unstable in biological samples orbuffered solutions.

There thus exists a need for assay methods that accurately detect and/orquantify vitamin D and vitamin D derivatives present in biologicalsamples in either free form or bound to DBP.

SUMMARY

Provided herein are antigenic molecules that can be used to generateantibodies capable of binding to vitamin D-derived molecules. In someembodiments, the antigenic molecule may be conjugated to a carrierprotein to form an antigenic compound. Carrier protein conjugation tothe antigenic molecule may occur through the use of a chemical linker.In some embodiments the antigenic protein may give rise to antibodies,such as monoclonal antibodies, that bind to different vitamin Dderivatives. Many of the described antigenic molecules incorporate avitamin D-C22 immunogen (FIG. 1( a)). More specifically, disclosedherein are vitamin D-C22 immunogens conjugated to bovine serum albumin(BSA) to produce vitamin D-C22 diaminobutane-suberoyl-BSA (vitamin D-C22BSA) (FIG. 1( c)). Alternatively, vitamin D-C22 may be conjugated to KLHto produce vitamin D-C22 diaminobutane-suberoyl-KLH (vit D-C22 KLH)(FIG. 1( d)). The described antigenic molecules and antigenic compoundsprovided herein may be used to produce antigen-reactive antibodies in amammal, such as a mouse. In turn, the immunized mammals may be used as asource of B cells to produce clonal hybridomas cell lines that produceantigen-reactive monoclonal antibodies.

Also disclosed herein are isolated antibodies and antigen-bindingfragments thereof, that can bind a vitamin D derivative, such as25-hydroxyvitamin D2 and/or 25-hydroxyvitamin D3, or a 25-hydroxyvitaminD analog, such as a vitamin D-C22 immunogenic molecule or compound, Insome embodiments the described antibodies are monoclonal antibodies. Insome aspects the described antibodies and antigen-binding fragments havea heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 10, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 11, aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 12, alight chain CDR1 having the amino acid sequence of SEQ ID NO: 26, alight chain CDR2 having the amino acid sequence of SEQ ID NO: 27, and alight chain CDR3 having the amino acid sequence of SEQ ID NO: 28. Suchan antibody is exemplified by the monoclonal antibody 10H9 that isdescribed herein. As will be further described, the antibodies providedherein, although monoclonal in nature, have the ability topreferentially bind more than one antigen. In this regard, some of theantibodies provided herein can bind to 25-hydroxyvitamin D2 and25-hydroxyvitamin D3 in an essentially indistinguishable manner. Alsoprovided are polynucleotides that encode the described antibodies andantigen-binding fragments and vectors for propagation and/or expressionof the described polynucleotides.

Further provided herein are methods for detecting vitamin D deficiencyin a subject by determining the level of total 25-hydroxyvitamin D in abiological sample derived from the subject wherein a decrease betweenthe level in the biological sample relative to the level in a normalcontrol or a threshold level of 30 ng/mL is indicative of a vitamin Ddeficiency in the subject.

Also provided herein are methods for treating a subject suspected ofhaving a vitamin D deficiency by determining the level of total25-hydroxyvitamin D in a biological sample derived from the subject and,in the event a decrease between the level in the biological samplerelative to the level in a normal control or a threshold level of 30ng/mL is detected, administering to the subject a treatment for vitaminD deficiency.

Also disclosed herein are methods for monitoring progression,regression, or stabilization of vitamin D deficiency in a subject inneed thereof by determining the level of total 25-hydroxyvitamin D in afirst biological sample derived from the subject at a first time andthen determining the level of total 25-hydroxyvitamin D in a secondbiological sample derived from the subject at a second time later thanthe first time wherein a decrease between the level in the firstbiological sample and the level in the second biological sample isindicative of progression or worsening of a vitamin D deficiency in thesubject, wherein little or no change between the level in the firstbiological sample and the level in the second biological sample isindicative of stabilization of a vitamin D deficiency in the subject,and wherein an increase between the level in the first biological sampleand the level in the second biological sample is indicative ofregression or improvement of a vitamin D deficiency in the subject.

Also provided herein are methods for monitoring treatment of vitamin Ddeficiency in a subject in need thereof by determining the level oftotal 25-hydroxyvitamin D in a first biological sample derived from thesubject at a first time and then determining the level of total25-hydroxyvitamin D in a second biological sample derived from thesubject at a second time later than the first time and followingtreatment of the subject for said vitamin D deficiency wherein anincrease in or stabilization of the level in the second biologicalsample relative to the level in the first biological sample isindicative of efficacy of the treatment of the vitamin D deficiency insaid subject, and wherein a decrease in the level in the secondbiological sample relative to the level in the first biological sampleis indicative of inefficacy of the treatment of the vitamin D deficiencyin said subject.

Methods for stabilizing 25-hydroxyvitamin D analogs by contacting the25-hydroxyvitamin D analog with 8-anilino-1-naphthalene sulfonate (ANS)are disclosed. The ANS may be in the form of ANS acid or a salt (e.g.,ANS sodium salt, ANS potassium salt, ANS hemimagnesium salt or ANSammonium salt).

Also provided herein are methods for detecting vitamin D deficiency in asubject. The methods for detecting vitamin D deficiency in a subjectinvolve determining the level of total 25-hydroxyvitamin D in abiological sample derived from the subject by combining the biologicalsample and a displacement buffer. In preferred embodiments, thedisplacement buffer contains 8-anilino-1-naphthalene sulfonate (ANS).The ANS may be in the form of ANS acid or a salt (e.g., ANS sodium salt,ANS potassium salt, ANS hemimagnesium salt or ANS ammonium salt). Thedisplacement buffer may further contain ethylene glycol. In someembodiments, the displacement buffer contains ANS and methanol. In somepreferred embodiments, the displacement buffer contains ANS, ethyleneglycol, and methanol. Next, an antibody or antigen-binding fragment thatpreferentially binds a vitamin D derivative, such as 25-hydroxyvitaminD2 and/or 25-hydroxyvitamin D3, or a 25-hydroxyvitamin D analog, such asa vitamin D-C22 immunogenic molecule or compound, conjugated to a firstlabel is combined with the assay mixture. The antibody orantigen-binding fragment that preferentially binds a vitamin Dderivative, such as 25-hydroxyvitamin D2 and/or 25-hydroxyvitamin D3, ora 25-hydroxyvitamin D analog, such as a vitamin D-C22 immunogenicmolecule or compound, is preferably an antibody or antigen-bindingfragment as described herein, for example, an antibody orantigen-binding fragment comprising a Lc CDR1 of SEQ ID NO: 26, a LcCDR2 of SEQ ID NO: 27, and a Lc CDR3 of SEQ ID NO: 28, a Hc CDR1 of SEQID NO: 10, a Hc CDR2 of SEQ ID NO: 11 and a Hc CDR3 of SEQ ID NO: 12. Inpreferred embodiments, the antibody is monoclonal antibody 10H9. Next a25-hydroxyvitamin D analog having a second label is combined with theassay mixture. The 25-hydroxyvitamin D analog can be present in astabilization buffer comprising 8-anilino-1-naphthalene sulfonate (ANS).The ANS may be in the form of ANS acid or a salt (e.g., ANS sodium salt,ANS potassium salt, ANS hemimagnesium salt or ANS ammonium salt). Insome embodiments, the 25-hydroxyvitamin D analog is conjugated to acarrier protein. A solid phase support conjugated to an antibody thatrecognizes the second label also is combined with the assay mixture. Thelevel of total 25-hydroxyvitamin D in the biological sample isdetermined by measuring the signal emitted by the first label, wherein areduced level of total 25-hydroxyvitamin D in the biological samplerelative to the level in a normal control or a threshold level of 30ng/mL is indicative of a vitamin D deficiency in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structure of vitamin D-C22 molecules andantigenic compounds. FIG. 1( a) depicts an unconjugated vitamin D-C22molecule. FIG. 1( b) provides a representation of a general vitaminD-C22 antigenic compound. FIGS. 1( c) and 1(d) show specific vitaminD-C22 antigenic compounds, where the vitamin D-C22 molecule isconjugated to either BSA or KLH by a linker: D-C22diaminobutane-suberoyl-BSA (FIG. 1( c)) and D-C22diaminobutane-suberoyl-KLH (FIG. 1( d)).

FIG. 2 shows a chemical process for producing vit D-C22 acid.

FIG. 3 depicts a chemical process for converting vit D-C22 acid toeither vitD-DAB-Suberoyl-NHS or vitD-DAB-PEG5-NHS.

FIG. 4 illustrates chemical reaction schemes for conjugating vit D-C22,vitD-DAB-Suberoyl-NHS or vitD-DAB-PEG5-NHS to a protein carrier.

FIG. 5 shows a two-step reaction for producing a vitaminD-DAB-PEG5-BSA-Fluorescein conjugate.

FIG. 6 provides a graphical representation of the degree of bindingbetween vitamin D-C22-Lc KLH and the antibody from the supernatant ofhybridoma 10H9 in the presence or absence of either 25-hydroxyvitamin D2or 25-hydroxyvitamin D3.

FIG. 7 is a graphical representation of the degree of binding betweenvitamin D C22-diaminobutane-suberoyl-alkaline phosphatase and purified10H9 monoclonal antibody in the presence or absence of either25-hydroxyvitamin D2 or 25-hydroxyvitamin D3.

FIG. 8 shows a schematic of the ADVIA Centaur Vitamin D Total Assay.This schematic is for illustrative purposes only and should not beconstrued as limiting in any way.

FIG. 9 shows the precision profile showing the limit of detection (LoD)and functional sensitivity (dashed line) of the ADVIA Centaur Vitamin DTotal assay.

FIG. 10 shows the correlation of Vitamin D Total levels collected from119 donors in serum red top and SST tubes and functional sensitivity.

FIG. 11 shows the correlation of Vitamin D Total levels collected from119 donors in serum red top and EDTA tubes.

FIG. 12 shows the correlation of the ADVIA Centaur and a commerciallyavailable Vitamin D Total assay.

FIG. 13 shows the correlation of the ADVIA Centaur and LC-MS/MS VitaminD Total assays.

FIG. 14 provides an annotated description of the 10H9 monoclonalantibody heavy chain variable region (SEQ ID NOS 33-34, respectively, inorder of appearance).

FIG. 15 provides an annotated description of the 10H9 monoclonalantibody light chain variable region (SEQ ID NOS 35-36, respectively, inorder of appearance).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various terms relating to aspects of the description are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinitions provided herein.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of up to ±20% from the specified value, as suchvariations are appropriate to perform the disclosed methods. Unlessotherwise indicated, all numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forthused in the specification and claims are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contain certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

“Isolated” means altered “by the hand of man” from the natural state. Ifa molecule or composition occurs in nature, it has been “isolated” if ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livingplant or animal is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated” as the term is employed herein.

“Polynucleotide,” synonymously referred to as “nucleic acid molecule” or“nucleic acids,” refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short nucleic acidchains, often referred to as oligonucleotides.

“Substantially the same” with respect to nucleic acid or amino acidsequences, means at least 65% identity between two or more sequences.Preferably, the term refers to at least 70% identity between two or moresequences, more preferably at least 75% identity, more preferably atleast 80% identity, more preferably at least 85% identity, morepreferably at least 90% identity, more preferably at least 91% identity,more preferably at least 92% identity, more preferably at least 93%identity, more preferably at least 94% identity, more preferably atleast 95% identity, more preferably at least 96% identity, morepreferably at least 97% identity, more preferably at least 98% identity,and more preferably at least 99% or greater identity. Such identity maybe determined using mBLAST algorithm (Altschul et al. (1990) Proc. Natl.Acad. Sci. USA 87:2264-8; Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-7).

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus inwhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

The term “operably linked” or “operably inserted” means that theregulatory sequences necessary for expression of the coding sequence areplaced in a nucleic acid molecule in the appropriate positions relativeto the coding sequence so as to enable expression of the codingsequence. By way of example, a promoter is operably linked with a codingsequence when the promoter is capable of controlling the transcriptionor expression of that coding sequence. Coding sequences may be operablylinked to promoters or regulatory sequences in a sense or antisenseorientation. The term “operably linked” is sometimes applied to thearrangement of other transcription control elements (e.g., enhancers) inan expression vector.

A cell has been “transformed” when exogenous or heterologous nucleicacids such as DNA have been introduced inside the cell. The transformingDNA may or may not be integrated (covalently linked) into the genome ofthe cell. In prokaryotes, yeast, and mammalian cells for example, thetransforming DNA may be maintained on an episomal element such as aplasmid. With respect to eukaryotic cells, a stably transformed cell, or“stable cell” is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the transforming DNA. A “clone” is a population ofcells derived from a single cell or common ancestor by mitosis. A “cellline” is a clone of a primary cell that is capable of stable growth invitro for many generations. In some examples provided herein, cells aretransformed by transfecting the cells with DNA.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts, monographs, andresearch literature. Modifications may occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. The same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide. Also, agiven polypeptide may contain many types of modifications. Polypeptidesmay be branched as a result of ubiquitination, and they may be cyclic,with or without branching. Cyclic, branched and branched cyclicpolypeptides may result from natural posttranslational processes or maybe made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination (See, for instance, Proteins—Structure and MolecularProperties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., Analysis for Protein Modifications and NonproteinCofactors, Meth Enzymol (1990) 182:626-646 and Rattan et al., ProteinSynthesis: Posttranslational Modifications and Aging, Ann NY Acad Sci(1992) 663:48-62).

“Biomolecules” include proteins, polypeptides, nucleic acids, lipids,monosaccharides, polysaccharides, and all fragments, analogs, homologs,conjugates, and derivatives thereof.

The terms “express” and “produce” are used synonymously herein, andrefer to the biosynthesis of a gene product. These terms encompass thetranscription of a gene into RNA. These terms also encompass translationof RNA into one or more polypeptides, and further encompass allnaturally occurring post-transcriptional and post-translationalmodifications. The expression or production of an antibody orantigen-binding fragment thereof may be within the cytoplasm of thecell, or into the extracellular milieu such as the growth medium of acell culture.

“Antibody” refers to all isotypes of immunoglobulins (IgG, IgA, IgE,IgM, IgD, and IgY) including various monomeric and polymeric forms ofeach isotype, unless otherwise specified.

An antigen-binding fragment is any proteinaceous structure that mayexhibit binding affinity for a particular antigen. Some antigen-bindingfragments are composed of portions of intact antibodies that retainantigen-binding specificity of the parent antibody molecule. Forexample, antigen-binding fragments may comprise at least one variableregion (either a heavy chain or light chain variable region) or one ormore CDRs of an antibody known to bind a particular antigen. Examples ofsuitable antigen-binding fragments include, without limitation diabodiesand single-chain molecules as well as Fab, F(ab′)2, Fc, Fabc, and Fvmolecules, single chain (Sc) antibodies, individual antibody lightchains, individual antibody heavy chains, chimeric fusions betweenantibody chains or CDRs and other proteins, protein scaffolds, ormolecules, heavy chain monomers or dimers, light chain monomers ordimers, dimers consisting of one heavy and one light chain, and thelike. All antibody isotypes may be used to produce antigen-bindingfragments. Additionally, antigen-binding fragments may includenon-antibody proteinaceous frameworks that may successfully incorporatepolypeptide segments in an orientation that confers affinity for a givenantigen of interest, such as protein scaffolds. Antigen-bindingfragments may be recombinantly produced or produced by enzymatic orchemical cleavage of intact antibodies. The phrase “an antibody orantigen-binding fragment thereof” may be used to denote that a givenantigen-binding fragment incorporates one or more amino acid segments ofthe antibody referred to in the phrase.

“Antibody compositions” refer to antibodies or binding fragments thereofthat are coupled with at least one pharmaceutically acceptable carrier,chemotherapeutic agent, or diagnostic moiety, as described herein.

“Specific binding” refers to the ability of an antibody, orantigen-binding fragment, to bind to a particular biomolecule with anaffinity that is greater than that with which it may bind otherbiomolecules. This term is used synonymously with “preferential”binding, meaning that a particular binding interaction is favored by theinteracting components over a majority of, but not all, other suchinteractions.

The embodiments described herein are not limited to particular methods,reagents, compounds, compositions or biological systems, which can, ofcourse, vary. Furthermore, the terminology used herein is for thepurpose of describing particular antibodies or antigen-binding fragmentsonly, and is not intended to be limiting.

Antigenic Molecules and Antigenic Compounds

Provided herein are antigenic molecules that can be used to generateantibodies capable of binding to vitamin D-derived molecules. In someembodiments, the antigenic molecule may be conjugated to a carrierprotein to form an antigenic compound. Carrier protein conjugation tothe antigenic molecule may occur through the use of a chemical linker.In some embodiments the antigenic protein may give rise to antibodiesthat bind equally to different vitamin D derivatives.

The antigenic molecules described herein are based on the use of avitamin D 22 carbon derivative (vitamin D-C22), which includes a C22carboxy group when unconjugated, as depicted in FIG. 1( a) (Hollis etal., Clin. Chem. 39(3):529-33 (1993)). Antigenic molecules based on thiscompound retain the common portion of 25-hydroxyvitamin D2 and25-hydroxyvitamin D3, as these molecules differ structurally by onlytheir side arms, which are absent from the vitamin D-C22 molecule. Giventhe structural commonalities between vitamin D-C22, 25-hydroxyvitamin D2and 25-hydroxyvitamin D3, antibodies generated using antigens based onvitamin D-C22 may be able to recognize both 25-hydroxyvitamin D2 and25-hydroxyvitamin D3.

The antigenic molecules disclosed herein may be combined with carrierproteins to produce antigenic compounds. The use of carrier proteins maybe useful to enhance the ability of the antigenic molecule to elicit animmune response in a mammal. For example carrier proteins may allow fora longer half-life in the host or allow for multiple antigenic moleculesto be attached to the same carrier, thus producing a multivalentantigenic compound. In some embodiments, the described antigenicmolecules may be affixed directly to the protein carrier. For example,vitamin D-C22 may be directly conjugated to bovine serum albumin (BSA).In some embodiments, a plurality of antigenic molecules may beconjugated to the same carrier protein to produce a multivalentantigenic compound. The number of antigenic molecules that may beconjugated to a given protein carrier will vary based on the carrierused. For example, BSA will accommodate the linkage of a relativelymodest number of antigenic molecules, perhaps about 10 to about 25;alternatively, a carrier such as keyhole limpet hemocyanin (KLH) mayaccommodate about 200 to about 300 antigenic molecules. In oneembodiment, vitamin D-C22 is conjugated to BSA such that from about 7 toabout 21 antigenic molecules are conjugated to the carrier. In oneembodiment, vitamin D-C22 is conjugated to BSA such that from about 12to about 16 antigenic molecules are conjugated to the carrier. Inanother such embodiment, about 14 vitamin D-C22 molecules are conjugatedto a BSA carrier. Those skilled in the art will understand that a widevariety of carrier proteins may be used for the purposes describedherein. Some suitable carriers include, KLH, PEGylated KLH, Concholepasconcholepas hemocyanin (CCH), cationized BSA, and ovalbumin to name onlya few. In some embodiments the antigenic molecules are conjugated to thecarrier protein via amine groups present on the carrier protein.Conjugation chemistry of this nature is commonly known to those skilledin the art.

Another aspect of the antigenic compounds described herein may be achemical linker that allows for indirect conjugation of a describedantigenic molecule to a described carrier protein. The chemical linkermay be comprised of alkyl, aryl, alkyloxy, amide, sulfonamide orcarbonyl or peptide groups. The conjugation of the vitamin D derivativeto the protein may be achieved by reaction between amino groups of theprotein and a reactive N-hydroxysuccinimide ester (NHS ester) group ofthe vitamin d derivative. The length of the linker may vary depending onthe carrier used and the number of antigenic molecules conjugated to thecarrier. In other embodiments, however, the same antigen molecule may beused with the same linker for conjugation to a variety of carrierproteins, such as BSA or KLH. In some embodiments, the linker iscomposed of a linear chain having the formula

where x and y can vary, independently, from about 1 to about 12. Thisprovides for a general antigenic compound having the formula depicted inFIG. 1( b). In some embodiments x may be 3 and y may be 6. In someembodiments x may be 3 and y may be 7. In some embodiments x may be 4and y may be 7. In some embodiments x may be 5 and y may be 7. In someembodiments x may be 4 and y may be 6. In some embodiments x may be 5and y may be 6. In some embodiments x may be 3 and y may be 5. In someembodiments x may be 4 and y may be 5. In some embodiments x may be 5and y may be 5. In some embodiments x may be 3 and y may be 4. In someembodiments x may be 4 and y may be 4. In some embodiments x may be 5and y may be 4. In some embodiments, the linker is used to conjugatevitamin D-C22 and BSA to produce vitamin D-C22diaminobutane-suberoyl-BSA (vit D-C22 BSA) (FIG. 1( c)). In anotherembodiment the linker is used to conjugate vitamin D-C22 and KLH toproduce vitamin D-C22 diaminobutane-suberoyl-KLH (vit D-C22 KLH) (FIG.1( d)).

The antigenic molecules and antigenic compounds provided herein may beused to produce antigen-reactive antibodies in a mammal Mammals that maybe used to produce antibodies include mouse, rat, goat, horse, swine,bovine, rabbit, donkey, human, and the like. In one embodiment, themammal is a mouse. Mammals having antigen positive sera may be used as asource of antibody producing B-cells that may be isolated and used toproduce long-lived antibody producing cells, such as hybridomas cells.In some embodiments, B cells isolated from immunized mice may be used toproduce hybridomas cells that produce antibodies that bind to vitaminD-C22 or vitamin D derivatives.

Antibodies

Described herein are isolated antibodies or antigen-binding fragmentsthat preferentially bind a vitamin D derivative, such as25-hydroxyvitamin D2 and/or 25-hydroxyvitamin D3, or a 25-hydroxyvitaminD analog, such as a vitamin D-C22 immunogenic molecule or compound. Inone embodiment, the antibody or antigen-binding fragment thereof is amonoclonal antibody or antigen-binding fragment.

There are five classes of immunoglobulins wherein the primary structureof the heavy chain, in the Fc region, determines the immunoglobulinclass. Specifically, the alpha, delta, epsilon, gamma, and mu chainscorrespond to IgA, IgD, IgE, IgG and IgM isotypes, respectively. Thedescribed antibodies or antigen-binding fragments include all isotypesand synthetic multimers of the four-chain immunoglobulin structure. Thedescribed antibodies or antigen-binding fragments also include the IgYisotype generally found in hen or turkey serum and hen or turkey eggyolk. Antibodies or antigen-binding fragments non-covalently,preferentially, and reversibly bind an antigen.

The antibodies or antigen-binding fragments of the disclosed subjectmatter may be derived from any species. For example, the antibodies orantigen-binding fragments may be derived from mouse, rat, goat, horse,swine, bovine, rabbit, donkey, human, and the like. In some embodimentsthe antibodies and antigen-binding fragments are derived from a mouse.In some embodiments the antibodies and antigen-binding fragments arederived from a mouse immunized with an immunogenic compound describedherein. In some embodiments the antibodies and antigen-binding fragmentsare derived from a mouse immunized with vitamin D-C22 BSA. In someembodiments the antibodies are monoclonal antibodies produced using theimmunogenic molecules or compounds described herein. The describedmonoclonal antibodies may be derived from a mouse immunized with any oneor more of the immunogenic molecules or compounds described herein. Forexample, a monoclonal antibody, or an antigen-binding fragment thereof,capable of preferentially binding to a vitamin D derivative, such as25-hydroxyvitamin D2 and/or 25-hydroxyvitamin D3, or a 25-hydroxyvitaminD analog, such as a vitamin D-C22 immunogenic molecule or compound maybe derived from a mouse immunized with a vitamin D-C22 immunogenicmolecule or compound.

In some embodiments, the antibodies or antigen-binding fragments may bechimeric. As used herein, the term “chimeric” antibody, orantigen-binding fragment, means an antibody, or antigen-binding fragmentthereof, having at least some portion of at least one variable domainderived from the antibody amino acid sequence of a non-human mammal, arodent, or a reptile, while the remaining portions of the antibody, orantigen-binding fragment thereof, are derived from a human. For example,a chimeric antibody may comprise a mouse antigen binding domain with ahuman Fc or other such structural domain.

In some embodiments the described antibodies may be humanized. Forexample, the CDRs of a human antibody may be replaced with the heavy andlight chain CDRs described herein to produce an antibody, orantigen-binding fragment thereof, that same the same or substantiallysimilar binding characteristics to the antibodies described herein, butis composed of human constant and framework regions. Methods forproducing such antibodies are commonly known to those skilled in the artand, thus should be considered to be within the scope of thisdisclosure.

The antibodies or antigen-binding fragments described herein may belabeled or otherwise conjugated to various chemical or biomoleculemoieties, for example, for diagnostic applications. The moieties may bedetectable labels, for example, chemiluminescent labels (e.g.,acridinium esters and sulfonamides, luminol and isoluminol),phosphorescent labels, fluorescent labels (e.g., FITC),electrochemiluminescent label (e.g, ruthenium (II) chelates), clonedenzyme donor, photosensitizer particle or chemiluminescer particle forluminescent oxygen channeling immunoassay (LOCI), lanthanide chelate fortime-resolved fluorescence immunoassay (TR-FIA), radiolabels, biotin,digoxigenin, enzymes and the like, for example, radionuclides, such as,but not limited to, tritium, carbon-14, lead-212, bismuth-212,astatine-211, iodine-131, scandium-47, rhenium-186, rhenium-188,yttrium-90, iodine-123, iodine-124, iodine-125, bromine-77, indium-111,and fissionable nuclides such as boron-10 or an actinide. In someembodiments enzymes may be conjugated to the described antibodies orantigen-binding proteins for the purposes of detecting bound antibody ina sample. Such enzyme conjugates include, but are not limited to,alkaline phosphatase (AP), horseradish peroxidase, beta-galactosidaseand glucose-6-phosphate dehydrogenase (G6PDH). Other enzymes used todetermine antibody binding in solution-based immunoassays would beunderstood by those skilled in the art to be suitable for use as aconjugate for the antibodies and antigen-binding fragments describedherein. In addition, compounds such as acridinium esters may also beconjugated to the provided antibodies and antigen-binding fragments toallow for detection in an immunoassay.

Antibody binding is primarily determined by the six CDR regions,especially H chain CDR3 (Kala et al., 132 J. Biochem. 535-41 (2002);Morea et al., 275 J. Mol. Biol. 269-94 (1998); and, Chothia et al., 196J. Mol. Biol. 901-17 (1987)). Antibody framework regions, however, canplay a role in antigen-antibody interactions (Panka et al., 85 Proc.Natl. Acad. Sci. USA 3080-4 (1988)), particularly in influencing theconformation of CDR loops (Foote et al., 224 J. Mol. Biol. 487-99(1992)). Thus, the described antibodies or antigen-binding fragments maycomprise any combination of H or L chain CDR or FWR regions that conferpreferential binding for 25-hydroxyvitamin D2 and/or 25-hydroxyvitaminD3 or vitamin D-C22-based immunogens. Domain shuffling experiments,which are routinely carried out in the art (Jirholt et al., 215 Gene471-6 (1998); Soderlind et al., 18 Nature Biotechnology 852-6 (2000)),may be employed to generate antibodies that preferentially bind25-hydroxyvitamin D2 and/or 25-hydroxyvitamin D3 or vitamin D-C22-basedimmunogens according to the specifications described and exemplifiedherein. Antibodies or antigen-binding fragments generated by such domainshuffling experiments are within the scope of the antibodies orantigen-binding fragments described herein. Furthermore, CDRs may alsobe arranged to bind a given antigen by engineering antibody-likeproteins to serve as CDR scaffolding (Nicaise et al., 13 Protein Sci.1882 (2004)). Such antigen-binding proteins are within the scope of theantibodies described herein.

The antibodies or antigen-binding fragments described herein can occurin a variety of forms, but will include one or more of the antibodysegments shown in Table 1.

TABLE 1 Antibody segments of the described antibodies andantigen-binding fragments thereof (“Lc” denotes light chain and “Hc”denotes heavy chain). 10H9 Antibody Amino Acid DNA Segment SEQ ID NO.SEQ ID NO. Lc CDR1 26 18 Lc CDR2 27 19 Lc CDR3 28 20 Lc FWR1 29 21 LcFWR2 30 22 Lc FWR3 31 23 Lc FWR4 25 17 Lc variable domain 32 24 Hc CDR110 2 Hc CDR2 11 3 Hc CDR3 12 4 Hc FWR1 13 5 Hc FWR2 14 6 Hc FWR3 15 7 HcFWR4 9 1 Hc variable domain 16 8

In some embodiments, the antibodies or antigen-binding fragments mayinclude a heavy chain CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 10. In some embodiments, the antibodiesor antigen-binding fragments may include a heavy chain CDR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 11. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 12. In some embodiments, antibodies orantigen-binding fragments may include a light chain CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 26. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 27. In some embodiments, antibodies orantigen-binding fragments may include a light chain CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 28. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 10; a CDR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 11; and a CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 12. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 26; a CDR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 27; and a CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 28. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain and a light chain, wherein the heavy chain has a CDR1 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 10;a CDR2 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 11; and a CDR3 amino acid sequence substantially the same as,or identical to, SEQ ID NO: 12; and the light chain has a CDR1 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 26;a CDR2 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 27; and a CDR3 amino acid sequence substantially the same as,or identical to, SEQ ID NO: 28. Antigen-binding arrangements of CDRs mayalso be engineered using antibody-like proteins as CDR scaffolding. Suchengineered antigen-binding proteins are within the scope of thedisclosure.

In some embodiments, antibodies or antigen-binding fragments may includea heavy chain FWR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 13. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain FWR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 14. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain FWR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 15. In some embodiments, antibodies orantigen-binding fragments may include a light chain FWR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 29. Insome embodiments, antibodies or antigen-binding fragments may include alight chain FWR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 30. In some embodiments, antibodies orantigen-binding fragments may include a light chain FWR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 31. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain having a FWR1 amino acid sequence that is substantially thesame as, or identical to, SEQ ID NO: 13; a FWR2 amino acid sequence thatis substantially the same as, or identical to, SEQ ID NO: 14; and a FWR3amino acid sequence that is substantially the same as, or identical to,SEQ ID NO: 15. In some embodiments, antibodies or antigen-bindingfragments may include a light chain having a FWR1 amino acid sequencethat is substantially the same as, or identical to, SEQ ID NO: 29; aFWR2 amino acid sequence that is substantially the same as, or identicalto, SEQ ID NO: 30; and a FWR3 amino acid sequence that is substantiallythe same as, or identical to, SEQ ID NO: 31. In some embodiments,antibodies or antigen-binding fragments may include a heavy chain and alight chain, wherein the heavy chain includes a FWR1 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 13; a FWR2 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 14;and a FWR3 amino acid sequence substantially the same as, or identicalto, SEQ ID NO: 15; and the light chain includes a FWR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 29; aFWR2 amino acid sequence substantially the same as, or identical to, SEQID NO: 30; and a FWR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 31.

In some embodiments, antibodies or antigen-binding fragments may includea heavy chain having a CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 10; a CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 11; and a CDR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 12; a FWR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 13; a FWR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 14; and a FWR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 15. Insome embodiments, the antibodies or antigen-binding fragments include alight chain having a CDR1 amino acid sequence substantially the same as,or identical to, SEQ ID NO: 26; a CDR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 27; and a CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 28; aFWR1 amino acid sequence substantially the same as, or identical to, SEQID NO: 29; a FWR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 30; and a FWR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 31. In someembodiments, the antibodies or antigen-binding fragments include a heavyand a light chain, wherein the heavy chain includes a CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 10; aCDR2 amino acid sequence substantially the same as, or identical to, SEQID NO: 11; and a CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 12; a FWR1 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 13; a FWR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 14; and a FWR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 15; and the light chain includes a CDR1 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 26; a CDR2 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 27;and a CDR3 amino acid sequence substantially the same as, or identicalto, SEQ ID NO: 28; a FWR1 amino acid sequence substantially the same as,or identical to, SEQ ID NO: 29; a FWR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 30; and a FWR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 31.Antigen-binding arrangements of CDRs and FWRs may also be engineeredusing antibody-like proteins as CDR scaffolding. Such engineeredantigen-binding proteins are within the scope of the disclosure.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein have a murine heavy chain. In some embodiments, theantibodies or antigen-binding fragments described herein have a murinelight chain. The described antibodies or antigen-binding fragments mayhave a heavy and a light chain, wherein the heavy chain is a murineheavy chain and the light chain is a murine light chain. In someembodiments, the antibodies or antigen-binding fragments describedherein have a murine IgG1 heavy chain. In some embodiments, theantibodies or antigen-binding fragments described herein have a murineIg kappa light chain. The described antibodies or antigen-bindingfragments may have a heavy and a light chain, wherein the heavy chain isa murine IgG1 heavy chain and the light chain is a murine Ig kappachain.

Polynucleotides Encoding the Antibodies

Also described are polynucleotides that encode antibodies orantigen-binding fragments that preferentially bind a vitamin Dderivative, such as 25-hydroxyvitamin D2 and/or 25-hydroxyvitamin D3, ora 25-hydroxyvitamin D analog, such as a vitamin D-C22 immunogenicmolecule or compound. The described polynucleotides can occur in avariety of forms and, therefore, may be native polynucleotides,recombinant polynucleotides (such as cDNA), or synthetically producedpolynucleotides. In some embodiments, the polynucleotides encode anantibody or antigen-binding fragment thereof having a heavy chain CDR1sequence substantially the same as, or identical to, SEQ ID NO: 10, forexample SEQ ID NO: 2. In some embodiments, the polynucleotides encode anantibody or antigen-binding fragment thereof having a heavy chain CDR2substantially the same as, or identical to, SEQ ID NO: 11, for exampleSEQ ID NO: 3. In some embodiments, the polynucleotides encode anantibody or antigen-binding fragment thereof having a heavy chain CDR3substantially the same as, or identical to, SEQ ID NO: 12, for exampleSEQ ID NO: 4. In some embodiments, the polynucleotides encode anantibody or antigen-binding fragment thereof having a light chain CDR1substantially the same as, or identical to, SEQ ID NO: 26, for exampleSEQ ID NO: 18. In some embodiments, the polynucleotides encode anantibody or antigen-binding fragment thereof having a light chain CDR2substantially the same as, or identical to, SEQ ID NO: 27, for exampleSEQ ID NO: 19. In some embodiments, the polynucleotides encode anantibody or antigen-binding fragment thereof having a light chain CDR3substantially the same as, or identical to, SEQ ID NO: 28, for exampleSEQ ID NO: 20. The polynucleotides may encode an antibody orantigen-binding fragment thereof having a heavy chain with a CDR1substantially the same as, or identical to, SEQ ID NO: 10, for exampleSEQ ID NO: 2; a CDR2 substantially the same as, or identical to, SEQ IDNO: 11, for example SEQ ID NO: 3; and a CDR3 substantially the same as,or identical to, SEQ ID NO: 12, for example SEQ ID NO: 4. Thepolynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain CDR1 substantially the same as, oridentical to, SEQ ID NO: 26, for example SEQ ID NO: 18; a CDR2substantially the same as, or identical to, SEQ ID NO: 27, for exampleSEQ ID NO: 19; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 28, for example SEQ ID NO: 20. The polynucleotides may encodean antibody or antigen-binding fragment thereof having a heavy chainCDR1 substantially the same as, or identical to, SEQ ID NO: 10, forexample SEQ ID NO: 2; a CDR2 encoded by a nucleotide sequencesubstantially the same as, or identical to, SEQ ID NO: 11, for exampleSEQ ID NO: 3; and a CDR3 encoded by a nucleotide sequence substantiallythe same as, or identical to, SEQ ID NO: 12, for example SEQ ID NO: 4;and a light chain CDR1 substantially the same as, or identical to, SEQID NO: 26, for example SEQ ID NO: 18; a CDR2 substantially the same as,or identical to, SEQ ID NO: 27, for example SEQ ID NO: 19; and a CDR3substantially the same as, or identical to, SEQ ID NO: 28, for exampleSEQ ID NO: 20.

In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR1 substantiallythe same as, or identical to, SEQ ID NO: 13, for example SEQ ID NO: 5.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR2 substantiallythe same as, or identical to, SEQ ID NO: 14, for example SEQ ID NO: 6.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR3 substantiallythe same as, or identical to, SEQ ID NO: 15, for example SEQ ID NO: 7.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain FWR1 substantiallythe same as, or identical to, SEQ ID NO: 29, for example SEQ ID NO: 21.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain FWR2 substantiallythe same as, or identical to, SEQ ID NO: 30, for example SEQ ID NO: 22.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain FWR3 substantiallythe same as, or identical to, SEQ ID NO: 31, for example SEQ ID NO: 23.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR1 substantiallythe same as, or identical to, SEQ ID NO: 13, for example SEQ ID NO: 5; aFWR2 substantially the same as, or identical to, SEQ ID NO: 14, forexample SEQ ID NO: 6; and a FWR3 substantially the same as, or identicalto, SEQ ID NO: 15, for example SEQ ID NO: 7. In some embodiments, thepolynucleotides encode an antibody or antigen-binding fragment thereofhaving a light FWR1 substantially the same as, or identical to, SEQ IDNO: 29, for example SEQ ID NO: 21; a FWR2 substantially the same as, oridentical to, SEQ ID NO: 30, for example SEQ ID NO: 22; and a FWR3substantially the same as, or identical to, SEQ ID NO: 31, for exampleSEQ ID NO: 23. In some embodiments, the polynucleotides encode anantibody or antigen-binding fragment thereof having a heavy chain and alight chain, wherein a heavy chain FWR1 is substantially the same as, oridentical to, SEQ ID NO: 13, for example SEQ ID NO: 5; a heavy chainFWR2 is substantially the same as, or identical to, SEQ ID NO: 14, forexample SEQ ID NO: 6; and a heavy chain FWR3 is substantially the sameas, or identical to, SEQ ID NO: 15, for example SEQ ID NO: 7; and alight chain FWR1 is substantially the same as, or identical to, SEQ IDNO: 29, for example SEQ ID NO: 21; a light chain FWR2 is substantiallythe same as, or identical to, SEQ ID NO: 30, for example SEQ ID NO: 22;and a light chain FWR3 substantially the same as, or identical to, SEQID NO: 31, for example SEQ ID NO: 23.

In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR1 substantiallythe same as, or identical to, SEQ ID NO: 10, for example SEQ ID NO: 2; aheavy chain CDR2 substantially the same as, or identical to, SEQ ID NO:11, for example SEQ ID NO: 3; and a heavy chain CDR3 substantially thesame as, or identical to, SEQ ID NO: 12, for example SEQ ID NO: 4; aheavy chain FWR1 substantially the same as, or identical to, SEQ ID NO:13, for example SEQ ID NO: 5; a heavy chain FWR2 substantially the sameas, or identical to, SEQ ID NO: 14, for example SEQ ID NO: 6; and aheavy chain FWR3 substantially the same as, or identical to, SEQ ID NO:15, for example SEQ ID NO: 7. In some embodiments, the polynucleotidesencode an antibody or antigen-binding fragment thereof having a lightchain CDR1 substantially the same as, or identical to, SEQ ID NO: 26,for example SEQ ID NO: 18; a light chain CDR2 substantially the same as,or identical to, SEQ ID NO: 27, for example SEQ ID NO: 19; and a lightchain CDR3 substantially the same as, or identical to, SEQ ID NO: 28,for example SEQ ID NO: 20; a light chain FWR1 substantially the same as,or identical to, SEQ ID NO: 29, for example SEQ ID NO: 21; a light chainFWR2 substantially the same as, or identical to, SEQ ID NO: 30, forexample SEQ ID NO: 22; and a light chain FWR3 substantially the same as,or identical to, SEQ ID NO: 31, for example SEQ ID NO: 23.

In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy and a light chain,wherein the polynucleotides encode a heavy chain CDR1 substantially thesame as, or identical to, SEQ ID NO: 10, for example SEQ ID NO: 2; aheavy chain CDR2 substantially the same as, or identical to, SEQ ID NO:11, for example SEQ ID NO: 3; a heavy chain CDR3 substantially the sameas, or identical to, SEQ ID NO: 12, for example SEQ ID NO: 4; a heavychain FWR1 substantially the same as, or identical to, SEQ ID NO: 13,for example SEQ ID NO: 5; a heavy chain FWR2 substantially the same as,or identical to, SEQ ID NO: 14, for example SEQ ID NO: 6; and a heavychain FWR3 substantially the same as, or identical to, SEQ ID NO: 15,for example SEQ ID NO: 7; and a light chain CDR1 substantially the sameas, or identical to, SEQ ID NO: 26, for example SEQ ID NO: 18; a lightchain CDR2 substantially the same as, or identical to, SEQ ID NO: 27,for example SEQ ID NO: 19; a light chain CDR3 substantially the same as,or identical to, SEQ ID NO: 28, for example SEQ ID NO: 20; a light chainFWR1 substantially the same as, or identical to, SEQ ID NO: 29, forexample SEQ ID NO: 21; a light chain FWR2 substantially the same as, oridentical to, SEQ ID NO: 30, for example SEQ ID NO: 22; and a lightchain FWR3 substantially the same as, or identical to, SEQ ID NO: 31,for example SEQ ID NO: 23.

Polynucleotides encoding engineered antigen-binding proteins also arewithin the scope of the disclosure.

In some embodiments, the polynucleotides described (and the peptidesthey encode) include a leader sequence. Any leader sequence known in theart may be employed. In some embodiments, the leader sequence may be, orbe based on, the heavy or light chain leader sequence of an antibody.The leader sequence may include, but is not limited to, a restrictionsite or a translation start site.

Because of the natural sequence variation likely to exist among heavyand light chains and the genes encoding them, one would expect to findsome level of variation within the amino acid sequences or the genesencoding the antibodies or antigen-binding fragments described herein,with little or no impact on their unique binding properties (e.g.,specificity and affinity). Such an expectation is due in part to thedegeneracy of the genetic code, as well as to the evolutionary successof conservative amino acid sequence variations, which do not appreciablyalter the nature of the encoded protein. Accordingly, some embodimentsinclude antibodies or antigen-binding fragments having 90%, 95%, 96%,97%, 98%, or 99% homology to the antibodies or antigen-binding fragmentsherein. Other embodiments include antibodies that preferentially bind avitamin D derivative, such as 25-hydroxyvitamin D2 and/or25-hydroxyvitamin D3, or a 25-hydroxyvitamin D analog, such as a vitaminD-C22 immunogenic molecule or compound, or antigen-binding fragments ofsuch antibodies, that have framework, scaffold, or other non-bindingregions that do not share significant homology with the antibodies andantigen-binding fragments described herein, but do incorporate one ormore CDRs or other sequences needed to confer binding that are 90%, 95%,96%, 97%, 98%, or 99% homologous to such sequences described herein.

The antibodies or antigen-binding fragments described herein includevariants having single or multiple amino acid substitutions, deletions,or additions that retain the biological properties (e.g., bindingaffinity or binding preference) of the described antibodies orantigen-binding fragments. The skilled person may produce variantshaving single or multiple amino acid substitutions, deletions, oradditions. These variants may include: (a) variants in which one or moreamino acid residues are substituted with conservative or nonconservativeamino acids, (b) variants in which one or more amino acids are added toor deleted from the polypeptide, (c) variants in which one or more aminoacids include a substituent group, and (d) variants in which thepolypeptide is fused with another peptide or polypeptide such as afusion partner, a protein tag or other chemical moiety, that may conferuseful properties to the polypeptide, such as, for example, an epitopefor an antibody, a polyhistidine sequence, a biotin moiety and the like.Antibodies or antigen-binding fragments described herein may includevariants in which amino acid residues from one species are substitutedfor the corresponding residue in another species, either at theconserved or nonconserved positions. In other embodiments, amino acidresidues at nonconserved positions are substituted with conservative ornonconservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques, are known to the person having ordinary skillin the art.

The antibodies or antigen-binding fragments described herein may embodyseveral antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. Antibodyor antigen-binding fragment thereof specificity is largely determined bythe amino acid sequence, and arrangement, of the CDRs. Therefore, theCDRs of one isotype may be transferred to another isotype withoutaltering antigen specificity. Alternatively, techniques have beenestablished to cause hybridomas to switch from producing one antibodyisotype to another (isotype switching) without altering antigenspecificity. Accordingly, such antibody isotypes are within the scope ofthe described antibodies or antigen-binding fragments.

The antibodies or antigen-binding fragments described herein havebinding affinities (in M) for a vitamin D derivative, such as25-hydroxyvitamin D2 and/or 25-hydroxyvitamin D3, or a 25-hydroxyvitaminD analog, such as a vitamin D-C22 immunogenic molecule or compound thatinclude a dissociation constant (K_(D)) of less than 1×10⁻². In someembodiments, the K_(D) is less than 1×10⁻³. In other embodiments, theK_(D) is less than 1×10⁻⁴. In some embodiments, the K_(D) is less than1×10⁻⁵. In still other embodiments, the K_(D) is less than 1×10⁻⁶,2×10⁻⁶, 3×10⁻⁶, 4×10⁻⁶, 5×10⁻⁶, 6×10⁻⁶, 7×10⁻⁶, 8×10⁻⁶, or 9×10⁻⁶. Inother embodiments, the K_(D) is less than 1×10⁻⁷, 2×10⁻⁷, or 3×10⁻⁷,2×10⁻⁷, 3×10⁻⁷, 4×10⁻⁷, 5×10⁻⁷, 6×10⁻⁷, 7×10⁻⁷, 8×10⁻⁷, or 9×10⁻⁷. Inother embodiments, the K_(D) is less than 1×10⁻⁸, 2×10⁻⁸, 3×10⁻⁸,4×10⁻⁸, 5×10⁻⁸, 6×10⁻⁸, 7×10⁻⁸, 8×10⁻⁸, or 9×10⁻⁸. In other embodiments,the K_(D) is less than 1×10⁻⁹, 2×10⁻⁹, 3×10⁻⁹, 4×10⁻⁹, 5×10⁻⁹, 6×10⁻⁹,7×10⁻⁹, 8×10⁻⁹, or 9×10. In other embodiments, the K_(D) is less than1×10⁻¹⁰, 2×10⁻¹⁰, 3×10⁻¹⁰, 2×10⁻¹⁰, 3×10⁻¹⁰, 4×10⁻¹⁰, 5×10⁻¹⁰, 6×10⁻¹⁰,7×10⁻¹⁰, 8×10⁻¹⁰, or 9×10⁻¹⁰. In still other embodiments, the K_(D) isless than 1×10⁻¹¹, 2×10⁻¹¹, 3×10⁻¹¹, 4×10⁻¹¹, 5×10⁻¹¹, 6×10⁻¹¹, 7×10⁻¹¹,8×10⁻¹¹, or 9×10⁻¹¹. In some embodiments, the K_(D) is less than1×10⁻¹². In other embodiments, the K_(D) is less than 1×10⁻¹³. In otherembodiments, the K_(D) is less than 1×10⁻¹⁴. In still other embodiments,the K_(D) is less than 1×10⁻¹⁵.

The antibodies or antigen-binding fragments described herein, in someembodiments, have equimolar recognition of 25-hydroxyvitamin D2 and25-hydroxyvitamin D3.

The antibodies or antigen-binding fragments described herein may bemodified, e.g., by the covalent attachment of any type of molecule tothe antibody or antigen-binding fragment thereof such that covalentattachment does not prevent the antibody or antigen-binding fragmentthereof from binding to its epitope. Examples of suitable modificationsinclude, but are not limited to glycosylation, acetylation, pegylation,phosphorylation, amidation, and the like. In some embodiments theantibodies or antigen-binding fragments may themselves be derivatized byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other proteins, and the like. The antibodies orantigen-binding fragments may have post-translational moieties thatimprove upon antibody or antigen-binding fragment thereof activity orstability. These moieties include sulfur, methyl, carbohydrate,phosphorus as well as other chemical groups commonly found onimmunoglobulin molecules. Furthermore, the antibodies or antigen-bindingfragments may contain one or more non-classical amino acids.

Antibodies or antigen-binding fragments described herein may be labeledwith or conjugated to diagnostic labels.

Also provided are vectors comprising the polynucleotides describedherein. The vectors can be expression vectors. Recombinant expressionvectors containing a sequence encoding a polypeptide of interest arethus provided. The expression vector may contain one or more additionalsequences such as but not limited to regulatory sequences (e.g.,promoter, enhancer), a selection marker, and a polyadenylation signal.In some embodiments, the vectors may encode the heavy chain segment ofan antibody or antigen-binding protein described herein. In someembodiments, the vectors may encode the light chain segment of anantibody or antigen-binding protein described herein. In some instancesthe heavy and light chain components may be encoded by a single vector.In other embodiments, the heavy and light chain components may beencoded by different vectors. Vectors for transforming a wide variety ofhost cells are well known and include, but are not limited to, plasmids,phagemids, cosmids, baculoviruses, bacmids, bacterial artificialchromosomes (BACs), yeast artificial chromosomes (YACs), as well asother bacterial, yeast and viral vectors.

Recombinant expression vectors within the scope of the descriptioninclude synthetic, genomic, or cDNA-derived nucleic acid fragments thatencode at least one recombinant protein which may be operably linked tosuitable regulatory elements. Such regulatory elements may include atranscriptional promoter, sequences encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. Expression vectors, especially mammalianexpression vectors, may also include one or more nontranscribed elementssuch as an origin of replication, a suitable promoter and enhancerlinked to the gene to be expressed, other 5′ or 3′ flankingnontranscribed sequences, 5′ or 3′ nontranslated sequences (such asnecessary ribosome binding sites), a polyadenylation site, splice donorand acceptor sites, or transcriptional termination sequences. An originof replication that confers the ability to replicate in a host may alsobe incorporated.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. Exemplary vectors may be constructed as described byOkayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

In some embodiments, the antibody or antigen-binding fragment-codingsequence is placed under control of a powerful constitutive promoter,such as the promoters for the following genes: hypoxanthinephosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase,beta-actin, human myosin, human hemoglobin, human muscle creatine, andothers. In addition, many viral promoters function constitutively ineukaryotic cells and are suitable for use with the describedembodiments. Such viral promoters include without limitation,Cytomegalovirus (CMV) immediate early promoter, the early and latepromoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, thelong terminal repeats (LTRs) of Maloney leukemia virus, HumanImmunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous SarcomaVirus (RSV), and other retroviruses, and the thymidine kinase promoterof Herpes Simplex Virus. In one embodiment, the antibody orantigen-binding fragment thereof coding sequence is placed under controlof an inducible promoter such as the metallothionein promoter,tetracycline-inducible promoter, doxycycline-inducible promoter,promoters that contain one or more interferon-stimulated responseelements (ISRE) such as protein kinase R 2′,5′-oligoadenylatesynthetases, Mx genes, ADAR1, and the like.

Vectors described herein may contain one or more Internal Ribosome EntrySite(s) (IRES). Inclusion of an IRES sequence into fusion vectors may bebeneficial for enhancing expression of some proteins. In someembodiments the vector system will include one or more polyadenylationsites (e.g., SV40), which may be upstream or downstream of any of theaforementioned nucleic acid sequences. Vector components may becontiguously linked, or arranged in a manner that provides optimalspacing for expressing the gene products (i.e., by the introduction of“spacer” nucleotides between the ORFs), or positioned in another way.Regulatory elements, such as the IRES motif, may also be arranged toprovide optimal spacing for expression.

The vectors may comprise selection markers, which are well known in theart. Selection markers include positive and negative selection markers,for example, antibiotic resistance genes (e.g., neomycin resistancegene, a hygromycin resistance gene, a kanamycin resistance gene, atetracycline resistance gene, a penicillin resistance gene), glutamatesythase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, orbacterial purine nucleoside phosphorylase gene for 6-methylpurineselection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). A nucleic acidsequence encoding a selection marker or the cloning site may be upstreamor downstream of a nucleic acid sequence encoding a polypeptide ofinterest or cloning site.

The vectors described herein may be used to transform various cells withthe genes encoding the described antibodies or antigen-bindingfragments. For example, the vectors may be used to generate antibody orantigen-binding fragment-producing cells. Thus, another aspect featureshost cells transformed with vectors comprising a nucleic acid sequenceencoding an antibody or antigen-binding fragment thereof that binds avitamin D derivative, such as 25-hydroxyvitamin D2 and/or25-hydroxyvitamin D3, or a 25-hydroxyvitamin D analog, such as a vitaminD-C22 immunogenic molecule or compound such as the antibodies orantigen-binding fragments described and exemplified herein.

Numerous techniques are known in the art for the introduction of foreigngenes into cells and may be used to construct the recombinant cells forpurposes of carrying out the described methods, in accordance with thevarious embodiments described and exemplified herein. The technique usedshould provide for the stable transfer of the heterologous gene sequenceto the host cell, such that the heterologous gene sequence is heritableand expressible by the cell progeny, and so that the necessarydevelopment and physiological functions of the recipient cells are notdisrupted. Techniques which may be used include but are not limited tochromosome transfer (e.g., cell fusion, chromosome mediated genetransfer, micro cell mediated gene transfer), physical methods (e.g.,transfection, spheroplast fusion, microinjection, electroporation,liposome carrier), viral vector transfer (e.g., recombinant DNA viruses,recombinant RNA viruses) and the like (described in Cline, 29 Pharmac.Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethyleneglycol (PEG)-induced fusion of bacterial protoplasts with mammaliancells may also be used to transform cells.

Cells suitable for use in the expression of the antibodies orantigen-binding fragments described herein are preferably eukaryoticcells, more preferably cells of plant, rodent, or human origin, forexample but not limited to NSO, CHO, perC.6, Tk-ts13, BHK, HEK293 cells,COS-7, T98G, CV-1/EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2/0 myelomacells, and BHK cell lines, among others. In addition, expression ofantibodies may be accomplished using hybridoma cells. Methods forproducing hybridomas are well established in the art.

Cells transformed with expression vectors described herein may beselected or screened for recombinant expression of the antibodies orantigen-binding fragments described herein. Recombinant-positive cellsare expanded and screened for subclones exhibiting a desired phenotype,such as high level expression, enhanced growth properties, or theability to yield proteins with desired biochemical characteristics, forexample, due to protein modification or altered post-translationalmodifications. These phenotypes may be due to inherent properties of agiven subclone or to mutation. Mutations may be effected through the useof chemicals, UV-wavelength light, radiation, viruses, insertionalmutagens, inhibition of DNA mismatch repair, or a combination of suchmethods.

Once a cell expressing the desired protein is identified, it can beexpanded and selected. Transformed cells may be selected in a number ofways. For example, cells may be selected for expression of thepolypeptide of interest. Cells transformed with a vector that contains aselectable marker, such as production of fluorescent protein, may bepositively selected for expression of the marker. In other embodiments,the cells containing a vector with a drug resistance gene may bepositively selected for the ability to grow under selective conditions.

Assays and Methods

The antibodies and antigen-binding fragments described herein may beused to detect vitamin D derivatives or analogues in a sample. In someembodiments, the antibodies and antigen-binding fragments are used todetect a vitamin D derivative, such as 25-hydroxyvitamin D2 and/or25-hydroxyvitamin D3, or a 25-hydroxyvitamin D analog, such as a vitaminD-C22 immunogenic molecule or compound. In some embodiments, thedescribed antibodies and antigen-binding fragments can be used to detectvitamin D derivatives or analogues in a biological sample obtained froma patient or a subject. In some embodiments the sample may be blood or ablood component, such as serum. In preferred embodiments, the patient orsubject is human. In some aspects the biological sample may be obtainedfrom a human patient or subject, for example, human blood. The describedmethods may be used with the antibodies and antigen-binding fragmentsalone, or in conjunction with other readily available antibodies ordetection reagents.

Provided herein are methods for detecting vitamin D deficiency in asubject. In preferred embodiments, the subject is human. The methodscomprise determining the level of total 25-hydroxyvitamin D in abiological sample derived from the subject wherein a decrease orreduction in level in the biological sample relative to the level in anormal control or to a threshold level of 30 ng/mL is indicative of avitamin D deficiency in the subject.

25-hydroxyvitamin D can be either of two forms, 25-hydroxyvitamin D2 and25-hydroxyvitamin D3. In preferred embodiments of the methods fordetecting vitamin D deficiency in a subject, the level of25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 is determined bycontacting the biological sample with an antibody or antigen-bindingfragment that recognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitaminD3.

Various heterogeneous and homogenous protocols, either competitive ornoncompetitive, can be employed in performing the methods for detectingvitamin D deficiency in a subject. In preferred embodiments, the methodsare performed by sequential competitive immunoassay. The Centaur™,Vista™, and Immulite™ are assay systems that can be used to perform acompetitive immunoassay.

In accordance with the methods for detecting vitamin D deficiency in asubject, the level of total 25-hydroxyvitamin D2 and 25-hydroxyvitaminD3 in a biological sample can be detected by enhanced chemiluminescence(ECL), enzyme immunoassay (EIA), immunohistochemistry (IHC), westernblot analysis, radioimmunoassay (RIA), immunofluorescence, equilibriumdialysis, immunodifferentiation, or enzyme-linked immunosorbant assay(ELISA).

In preferred embodiments of the methods for detecting vitamin Ddeficiency in a subject, the antibody or antigen-binding fragment usedin accordance with the methods is not cross-reactive with vitamin D2and/or vitamin D3. In preferred embodiments, the antibody orantigen-binding fragment is an antibody or antigen-binding fragment asdescribed herein. For example, the antibody or antigen-binding fragmentcomprises a Lc CDR1 of SEQ ID NO: 26, a Lc CDR2 of SEQ ID NO: 27, and aLc CDR3 of SEQ ID NO: 28, a Hc CDR1 of SEQ ID NO: 10, a Hc CDR2 of SEQID NO: 11 and a Hc CDR3 of SEQ ID NO: 12. In some embodiments, theantibody or antigen-binding fragment has the property of equimolarrecognition for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. Inpreferred embodiments, the antibody is monoclonal antibody 10H9.

Antibodies and antigen-binding fragments that recognize25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 that can be used in themethods for detecting vitamin D deficiency in a subject can be labeled,for example, with a detectable label. Exemplary labels include but arenot limited to chemiluminescent compounds (e.g., an acridinium estercompound), a phosphorescent compound, a fluorescent compound, aradiolabel, biotin, or an enzyme. The mentioned exemplary labels canusually only be detected when excited by methods that include but arenot limited to addition of different chemicals, stimulation by light orexposure to substrate or other compounds. When using acridinium estercompound, chemiluminescence is triggered by peroxide and acid/baseresulting in a flash that can be read by appropriate instrumentation. Anoptional wash step may be used before initiating detectability of thedetectable label.

The antibody or antigen-binding fragment can be immobilized on a solidphase support.

The antibody or antigen-binding fragment can be conjugated to a carrierprotein. The complex between the antibody or antigen-binding protein andcarrier protein also can be immobilized on a solid phase support.

Solid phase supports for use in the methods for detecting vitamin Ddeficiency in a subject include paramagnetic particles; cross-linkeddextran available under the trademark SEPHADEX (Pharmacia FineChemicals, Piscataway, N.J.); agarose; polystyrene beads; polyvinylchloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- ornylon-based webs such as sheets, strips or paddles; or tubes, plates orthe wells of a microtiter plate such as those made from polystyrene orpolyvinylchloride. When using paramagnetic particles, some source of amagnetic field may be used to retain the particles and molecules bounddirectly or indirectly to the particles during an optional wash step.The molecules may be bound covalently, by salt-bridges, hydrogen bondingor another type of bond.

The biological sample can be blood, blood serum or blood plasma. In someembodiments, the biological sample may be stored under biologicalconditions for up to 24 hours prior to use in the methods describedherein.

In some embodiments of the methods for detecting vitamin D deficiency ina subject, the biological sample is treated or combined with8-anilino-1-naphthalene sulfonate (ANS) before contacting the biologicalsample with an antibody or antigen-binding fragment that recognizes both25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 Alternatively, thebiological sample can be treated or combined with8-anilino-1-naphthalene sulfonate (ANS) concurrently with contacting thebiological sample with an antibody or antigen-binding fragment thatrecognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The ANSmay be in the form of ANS acid or a salt (e.g., ANS sodium salt, ANSpotassium salt, ANS hemimagnesium salt or ANS ammonium salt). The ANSmay be present, for example, in a displacement buffer. Methanol mayoptionally be used with the 8-anilino-1-naphthalene sulfonate (ANS)either before contacting the biological sample with an antibody orantigen-binding fragment or concurrently with contacting the biologicalsample with an antibody or antigen-binding fragment. Methanol may, forexample, be included in the displacement buffer.

In some embodiments of the methods for detecting vitamin D deficiency ina subject, the biological sample also is treated or combined with8-anilino-1-naphthalene sulfonate (ANS) and ethylene glycol beforecontacting the biological sample with an antibody that recognizes both25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 or antigen-bindingfragment. Alternatively, the biological sample can be treated orcombined with 8-anilino-1-naphthalene sulfonate (ANS) and ethyleneglycol concurrently with contacting the biological sample with anantibody that recognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitaminD3 or antigen-binding fragment. The ANS may be in the form of ANS acidor a salt (e.g., ANS sodium salt, ANS potassium salt, ANS hemimagnesiumsalt or ANS ammonium salt). The ANS and ethylene glycol may be presentin a displacement buffer. Methanol may optionally be used with the8-anilino-1-naphthalene sulfonate (ANS) and ethylene glycol eitherbefore contacting the biological sample with an antibody orantigen-binding fragment or concurrently with contacting the biologicalsample with an antibody or antigen-binding fragment. Methanol may, forexample, be included in the displacement buffer.

In some embodiments of the methods for detecting vitamin D deficiency ina subject, a 25-hydroxyvitamin D analog is added to the biologicalsample following the contacting step. The 25-hydroxyvitamin D analog canbe labeled. 25-Hydroxyvitamin D analog or labeled 25-hydroxyvitamin Danalog can also be present in a stabilization buffer comprising8-anilino-1-naphthalene sulfonate (ANS). The ANS may be in the form ofANS acid or a salt (e.g., ANS sodium salt, ANS potassium salt, ANShemimagnesium salt or ANS ammonium salt).

The vitamin D analogs described herein may be based on the use of avitamin D 22 carbon derivative (vitamin D-C22), which includes a C22carboxy group when unconjugated, as depicted in FIG. 1( a) (Hollis etal., Clin. Chem. 39(3):529-33 (1993)). In some embodiments, the vitaminD analog may be vitamin D-C22. In some embodiments, the vitamin D analogmay be conjugated to a carrier protein.

In some embodiments of the methods for detecting vitamin D deficiency ina subject, the described vitamin D analogs may be affixed directly tothe protein carrier. For example, vitamin D-C22 may be directlyconjugated to bovine serum albumin (BSA). The number of vitamin Danalogs that may be conjugated to a given protein carrier will varybased on the carrier used. Those skilled in the art will understand thata wide variety of carrier proteins may be used for the purposesdescribed herein. Some suitable carriers include, KLH, PEGylated KLH,Concholepas concholepas hemocyanin (CCH), cationized BSA, and ovalbuminto name only a few.

Carrier protein conjugation to the vitamin D analog may occur throughthe use of a chemical linker. The chemical linker may be comprised ofalkyl, aryl, alkyloxy, amide, sulfonamide or carbonyl or peptide groups.The conjugation of the vitamin D analog or vitamin D derivative to theprotein may be achieved by reaction between amino groups of the proteinand a reactive N-hydroxysuccinimide ester (NHS ester) group of thevitamin D analog or vitamin D derivative.

In some embodiments of the methods for detecting vitamin D deficiency ina subject, vitamin D levels can be detected in 20 minutes or lessstarting from the point at which the biological sample is combined withthe displacement buffer.

Vitamin D deficiency in the subject can be indicative of or associatedwith a disease. Diseases associated with vitamin D deficiency caninclude: rickets, osteomalacia, high blood pressure, osteoporosis,autoimmune disease, cardiovascular disease, schizophrenia, depression,nervous system disease, diabetes, infectious disease, asthma, allergy orcancer.

Also provided herein are methods for treating a subject suspected ofhaving a vitamin D deficiency by determining the level of total25-hydroxyvitamin D in a biological sample derived from the subject and,if a decrease between the level in the biological sample relative to thelevel in a normal control or a threshold level of 30 ng/mL isdetermined, administering to the subject a treatment for vitamin Ddeficiency. In preferred embodiments, the subject is human. There areseveral suitable ways to treat a vitamin D deficiency. Vitamin Ddeficiency can be treated by supplementing vitamin D intake or byphototherapy. Phototherapy can include increased exposure to naturalsunlight or exposure to artificial sources of ultraviolet B light.

In preferred embodiments of the methods for treating a subject suspectedof having a vitamin D deficiency, the level of 25-hydroxyvitamin D2 and25-hydroxyvitamin D3 is determined by contacting the biological samplewith an antibody or antigen-binding fragment that recognizes both25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

Various heterogeneous and homogenous protocols, either competitive ornoncompetitive, can be employed in performing the methods for treating asubject suspected of having a vitamin D deficiency. In preferredembodiments, the methods are performed by sequential competitiveimmunoassay. The Centaur™, Vista™, and Immulite™ are assay systems thatcan be used to perform a competitive immunoassay.

In accordance with the methods for treating a subject suspected ofhaving a vitamin D deficiency, the level of total 25-hydroxyvitamin D2and 25-hydroxyvitamin D3 in a biological sample can be detected byenhanced chemiluminescence (ECL), enzyme immunoassay (EIA),immunohistochemistry (IHC), western blot analysis, radioimmunoassay(RIA), immunofluorescence, equilibrium dialysis, immunodifferentiation,or enzyme-linked immunosorbant assay (ELISA).

In preferred embodiments of the methods for treating a subject suspectedof having a vitamin D deficiency, the antibody or antigen-bindingfragment used in accordance with the methods is not cross-reactive withvitamin D2 and/or vitamin D3. In preferred embodiments, the antibody orantigen-binding fragment is an antibody or antigen-binding fragment asdescribed herein. For example, the antibody or antigen-binding fragmentcomprises a Lc CDR1 of SEQ ID NO: 26, a Lc CDR2 of SEQ ID NO: 27, and aLc CDR3 of SEQ ID NO: 28, a Hc CDR1 of SEQ ID NO: 10, a Hc CDR2 of SEQID NO: 11 and a Hc CDR3 of SEQ ID NO: 12. In some embodiments, theantibody or antigen-binding fragment has the property of equimolarrecognition for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. Inpreferred embodiments, the antibody is monoclonal antibody 10H9.

Antibodies and antigen-binding fragments that recognize25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 that can be used in themethods for treating a subject suspected of having a vitamin Ddeficiency can be labeled, for example, with a detectable label.Exemplary labels include but are not limited to chemiluminescentcompounds (e.g., an acridinium ester compound), a phosphorescentcompound, a fluorescent compound, a radiolabel, biotin, or an enzyme.The mentioned exemplary labels can usually only be detected when excitedby methods that include but are not limited to addition of differentchemicals, stimulation by light or exposure to substrate or othercompounds. When using acridinium ester compound, chemiluminescence istriggered by peroxide and acid/base resulting in a flash that can beread by appropriate instrumentation. An optional wash step may be usedbefore initiating detectability of the detectable label.

The antibody or antigen-binding fragment can be immobilized on a solidphase support.

The antibody or antigen-binding fragment can be conjugated to a carrierprotein. The complex between the antibody or antigen-binding protein andcarrier protein also can be immobilized on a solid phase support.

Solid phase supports for use in the methods described herein includeparamagnetic particles; cross-linked dextran available under thetrademark SEPHADEX (Pharmacia Fine Chemicals, Piscataway, N.J.);agarose; polystyrene beads; polyvinyl chloride, polystyrene,cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such assheets, strips or paddles; or tubes, plates or the wells of a microtiterplate such as those made from polystyrene or polyvinylchloride. Whenusing paramagnetic particles, some source of a magnetic field may beused to retain the particles and molecules bound directly or indirectlyto the particles during an optional wash step. The molecules may bebound covalently, by salt-bridges, hydrogen bonding or another type ofbond.

The biological sample can be blood, blood serum or blood plasma. In someembodiments, the biological sample may be stored under biologicalconditions for up to 24 hours prior to use in the methods describedherein.

In some embodiments of the methods for treating a subject suspected ofhaving a vitamin D deficiency, the biological sample is treated orcombined with 8-anilino-1-naphthalene sulfonate (ANS) before contactingthe biological sample with an antibody or antigen-binding fragment thatrecognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3Alternatively, the biological sample can be treated or combined with8-anilino-1-naphthalene sulfonate (ANS) concurrently with contacting thebiological sample with an antibody or antigen-binding fragment thatrecognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The ANSmay be in the form of ANS acid or a salt (e.g., ANS sodium salt, ANSpotassium salt, ANS hemimagnesium salt or ANS ammonium salt). The ANSmay be present, for example, in a displacement buffer. Methanol mayoptionally be used with the 8-anilino-1-naphthalene sulfonate (ANS)either before contacting the biological sample with an antibody orantigen-binding fragment or concurrently with contacting the biologicalsample with an antibody or antigen-binding fragment. Methanol may, forexample, be included in the displacement buffer.

In some embodiments of the methods for treating a subject suspected ofhaving a vitamin D deficiency, the biological sample also is treated orcombined with 8-anilino-1-naphthalene sulfonate (ANS) and ethyleneglycol before contacting the biological sample with an antibody thatrecognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 orantigen-binding fragment. Alternatively, the biological sample can betreated or combined with 8-anilino-1-naphthalene sulfonate (ANS) andethylene glycol concurrently with contacting the biological sample withan antibody that recognizes both 25-hydroxyvitamin D2 and25-hydroxyvitamin D3 or antigen-binding fragment. The ANS may be in theform of ANS acid or a salt (e.g., ANS sodium salt, ANS potassium salt,ANS hemimagnesium salt or ANS ammonium salt). The ANS and ethyleneglycol may be present in a displacement buffer. Methanol may optionallybe used with the 8-anilino-1-naphthalene sulfonate (ANS) and ethyleneglycol either before contacting the biological sample with an antibodyor antigen-binding fragment or concurrently with contacting thebiological sample with an antibody or antigen-binding fragment. Methanolmay, for example, be included in the displacement buffer.

In some embodiments of the methods for treating a subject suspected ofhaving a vitamin D deficiency, a 25-hydroxyvitamin D analog is added tothe biological sample following the contacting step. The25-hydroxyvitamin D analog can be labeled. 25-Hydroxyvitamin D analog orlabeled 25-hydroxyvitamin D analog can also be present in astabilization buffer comprising 8-anilino-1-naphthalene sulfonate (ANS).The ANS may be in the form of ANS acid or a salt (e.g., ANS sodium salt,ANS potassium salt, ANS hemimagnesium salt or ANS ammonium salt).

The vitamin D analogs described herein may be based on the use of avitamin D 22 carbon derivative (vitamin D-C22), which includes a C22carboxy group when unconjugated, as depicted in FIG. 1( a) (Hollis etal., Clin. Chem. 39(3):529-33 (1993)). In some embodiments, the vitaminD analog may be vitamin D-C22. In some embodiments, the vitamin D analogmay be conjugated to a carrier protein.

In some embodiments of the methods for treating a subject suspected ofhaving a vitamin D deficiency, the described vitamin D analogs may beaffixed directly to the protein carrier. For example, vitamin D-C22 maybe directly conjugated to bovine serum albumin (BSA). The number ofvitamin D analogs that may be conjugated to a given protein carrier willvary based on the carrier used. Those skilled in the art will understandthat a wide variety of carrier proteins may be used for the purposesdescribed herein. Some suitable carriers include, KLH, PEGylated KLH,Concholepas concholepas hemocyanin (CCH), cationized BSA, and ovalbuminto name only a few.

Carrier protein conjugation to the vitamin D analog may occur throughthe use of a chemical linker. The chemical linker may be comprised ofalkyl, aryl, alkyloxy, amide, sulfonamide or carbonyl or peptide groups.The conjugation of the vitamin D analog or vitamin D derivative to theprotein may be achieved by reaction between amino groups of the proteinand a reactive N-hydroxysuccinimide ester (NHS ester) group of thevitamin D analog or vitamin D derivative.

In some embodiments of the methods for treating a subject suspected ofhaving a vitamin D deficiency, vitamin D levels can be detected in 20minutes or less starting from the point at which the biological sampleis combined with the displacement buffer.

Vitamin D deficiency in the subject can be indicative of or associatedwith a disease. Diseases associated with vitamin D deficiency caninclude: rickets, osteomalacia, high blood pressure, osteoporosis,autoimmune disease, cardiovascular disease, schizophrenia, depression,nervous system disease, diabetes, infectious disease, asthma, allergy orcancer.

Further provided herein are methods for monitoring progression ofvitamin D deficiency in a subject in need thereof by determining thelevel of total 25-hydroxyvitamin D in a first biological sample derivedfrom the subject at a first time and then determining the level of total25-hydroxyvitamin D in a second biological sample derived from thesubject at a second time later than the first time wherein a decreasebetween the level in the first biological sample and the level in thesecond biological sample is indicative of the progression of a vitamin Ddeficiency in the subject, wherein little or no change between the levelin the first biological sample and the level in the second biologicalsample is indicative of stabilization of a vitamin D deficiency in thesubject, and wherein an increase between the level in the firstbiological sample and the level in the second biological sample isindicative of regression of a vitamin D deficiency in the subject. Inpreferred embodiments, the subject is human.

In preferred embodiments of the methods for monitoring progression ofvitamin D deficiency in a subject, the level of 25-hydroxyvitamin D2 and25-hydroxyvitamin D3 is determined by contacting the biological samplewith an antibody or antigen-binding fragment that recognizes both25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

Various heterogeneous and homogenous protocols, either competitive ornoncompetitive, can be employed in performing the methods for monitoringprogression of vitamin D deficiency in a subject. In preferredembodiments, the methods are performed by competitive immunoassay. TheCentaur™, Vista™, and Immulite™ are assay systems that can be used toperform a competitive immunoassay.

In accordance with the methods for monitoring progression of vitamin Ddeficiency in a subject, the level of total 25-hydroxyvitamin D2 and25-hydroxyvitamin D3 in a biological sample can be detected by enhancedchemiluminescence (ECL), enzyme immunoassay (EIA), immunohistochemistry(IHC), western blot analysis, radioimmunoassay (RIA),immunofluorescence, equilibrium dialysis, immunodifferentiation, orenzyme-linked immunosorbant assay (ELISA).

In preferred embodiments of the methods for monitoring progression ofvitamin D deficiency in a subject, the antibody or antigen-bindingfragment used in accordance with the methods is not cross-reactive withvitamin D2 and/or vitamin D3. In preferred embodiments, the antibody orantigen-binding fragment is an antibody or antigen-binding fragment asdescribed herein. For example, the antibody or antigen-binding fragmentcomprises a Lc CDR1 of SEQ ID NO: 26, a Lc CDR2 of SEQ ID NO: 27, and aLc CDR3 of SEQ ID NO: 28, a Hc CDR1 of SEQ ID NO: 10, a Hc CDR2 of SEQID NO: 11 and a Hc CDR3 of SEQ ID NO: 12. In some embodiments, theantibody or antigen-binding fragment has the property of equimolarrecognition for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. Inpreferred embodiments, the antibody is monoclonal antibody 10H9.

Antibodies and antigen-binding fragments that recognize25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 that can be used in thesemethods can be labeled, for example, with a detectable label. Exemplarylabels include but are not limited to chemiluminescent compounds (e.g.,an acridinium ester compound), a phosphorescent compound, a fluorescentcompound, a radiolabel, biotin, or an enzyme. The mentioned exemplarylabels can usually only be detected when excited by methods that includebut are not limited to addition of different chemicals, stimulation bylight or exposure to substrate or other compounds. When using acridiniumester compound, chemiluminescence is triggered by peroxide and acid/baseresulting in a flash that can be read by appropriate instrumentation. Anoptional wash step may be used before initiating detectability of thedetectable label.

The antibody or antigen-binding fragment can be immobilized on a solidphase support.

The antibody or antigen-binding fragment can be conjugated to a carrierprotein. The complex between the antibody or antigen-binding protein andcarrier protein also can be immobilized on a solid phase support.

Solid phase supports for use in the methods for monitoring progressionof vitamin D deficiency in a subject include paramagnetic particles;cross-linked dextran available under the trademark SEPHADEX (PharmaciaFine Chemicals, Piscataway, N.J.); agarose; polystyrene beads; polyvinylchloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- ornylon-based webs such as sheets, strips or paddles; or tubes, plates orthe wells of a microtiter plate such as those made from polystyrene orpolyvinylchloride. When using paramagnetic particles, some source of amagnetic field may be used to retain the particles and molecules bounddirectly or indirectly to the particles during an optional wash step.The molecules may be bound covalently, by salt-bridges, hydrogen bondingor another type of bond.

The biological sample can be blood, blood serum or blood plasma. In someembodiments, the biological sample may be stored under biologicalconditions for up to 24 hours prior to use in the methods describedherein.

In some embodiments of the methods for monitoring progression of vitaminD deficiency in a subject, the biological sample is treated or combinedwith 8-anilino-1-naphthalene sulfonate (ANS) before contacting thebiological sample with an antibody or antigen-binding fragment thatrecognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3Alternatively, the biological sample can be treated or combined with8-anilino-1-naphthalene sulfonate (ANS) concurrently with contacting thebiological sample with an antibody or antigen-binding fragment thatrecognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The ANSmay be in the form of ANS acid or a salt (e.g., ANS sodium salt, ANSpotassium salt, ANS hemimagnesium salt or ANS ammonium salt). The ANSmay be present, for example, in a displacement buffer. Methanol mayoptionally be used with the 8-anilino-1-naphthalene sulfonate (ANS)either before contacting the biological sample with an antibody orantigen-binding fragment or concurrently with contacting the biologicalsample with an antibody or antigen-binding fragment. Methanol may, forexample, be included in the displacement buffer.

In some embodiments of the methods for monitoring progression of vitaminD deficiency in a subject, the biological sample also is treated orcombined with 8-anilino-1-naphthalene sulfonate (ANS) and ethyleneglycol before contacting the biological sample with an antibody thatrecognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 orantigen-binding fragment. Alternatively, the biological sample can betreated or combined with 8-anilino-1-naphthalene sulfonate (ANS) andethylene glycol concurrently with contacting the biological sample withan antibody that recognizes both 25-hydroxyvitamin D2 and25-hydroxyvitamin D3 or antigen-binding fragment. The ANS may be in theform of ANS acid or a salt (e.g., ANS sodium salt, ANS potassium salt,ANS hemimagnesium salt or ANS ammonium salt). The ANS and ethyleneglycol may be present in a displacement buffer. Methanol may optionallybe used with the 8-anilino-1-naphthalene sulfonate (ANS) and ethyleneglycol either before contacting the biological sample with an antibodyor antigen-binding fragment or concurrently with contacting thebiological sample with an antibody or antigen-binding fragment. Methanolmay, for example, be included in the displacement buffer.

In some embodiments of the methods for monitoring progression of vitaminD deficiency in a subject, a 25-hydroxyvitamin D analog is added to thebiological sample following the contacting step. The 25-hydroxyvitamin Danalog can be labeled. 25-Hydroxyvitamin D analog or labeled25-hydroxyvitamin D analog can also be present in a stabilization buffercomprising 8-anilino-1-naphthalene sulfonate (ANS). The ANS may be inthe form of ANS acid or a salt (e.g., ANS sodium salt, ANS potassiumsalt, ANS hemimagnesium salt or ANS ammonium salt).

The vitamin D analogs described herein may be based on the use of avitamin D 22 carbon derivative (vitamin D-C22), which includes a C22carboxy group when unconjugated, as depicted in FIG. 1( a) (Hollis etal., Clin. Chem. 39(3):529-33 (1993)). In some embodiments, the vitaminD analog may be vitamin D-C22. In some embodiments, the vitamin D analogmay be conjugated to a carrier protein.

In some embodiments of the methods for monitoring progression of vitaminD deficiency in a subject, the described vitamin D analogs may beaffixed directly to the protein carrier. For example, vitamin D-C22 maybe directly conjugated to bovine serum albumin (BSA). The number ofvitamin D analogs that may be conjugated to a given protein carrier willvary based on the carrier used. Those skilled in the art will understandthat a wide variety of carrier proteins may be used for the purposesdescribed herein. Some suitable carriers include, KLH, PEGylated KLH,Concholepas concholepas hemocyanin (CCH), cationized BSA, and ovalbuminto name only a few.

Carrier protein conjugation to the vitamin D analog may occur throughthe use of a chemical linker. The conjugation of the vitamin D analog orvitamin D derivative to the protein may be achieved by reaction betweenamino groups of the protein and a reactive N-hydroxysuccinimide ester(NHS ester) group of the vitamin D analog or vitamin D derivative.

In some embodiments of the methods for monitoring progression of vitaminD deficiency in a subject, vitamin D levels can be detected in 20minutes or less starting from the point at which the biological sampleis combined with the displacement buffer.

Vitamin D deficiency in the subject can be indicative of or associatedwith a disease. Diseases associated with vitamin D deficiency caninclude: rickets, osteomalacia, high blood pressure, osteoporosis,autoimmune disease, cardiovascular disease, schizophrenia, depression,nervous system disease, diabetes, infectious disease, asthma, allergy orcancer.

Also provided herein are methods for monitoring treatment of vitamin Ddeficiency in a subject in need thereof by determining the level oftotal 25-hydroxyvitamin D in a first biological sample derived from thesubject at a first time and then determining the level of total25-hydroxyvitamin D in a second biological sample derived from thesubject at a second time later than the first time and followingtreatment of the subject for said vitamin D deficiency wherein anincrease in or stabilization of the level in the second biologicalsample relative to the level in the first biological sample isindicative of efficacy of the treatment of the vitamin D deficiency insaid subject, and wherein a decrease in the level in the secondbiological sample relative to the level in the first biological sampleis indicative of inefficacy of the treatment of the vitamin D deficiencyin said subject. In preferred embodiments, the subject is human.

In preferred embodiments of the methods for monitoring treatment ofvitamin D deficiency in a subject, the level of 25-hydroxyvitamin D2 and25-hydroxyvitamin D3 is determined by contacting the biological samplewith an antibody or antigen-binding fragment that recognizes both25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

Various heterogeneous and homogenous protocols, either competitive ornoncompetitive, can be employed in performing the methods for monitoringtreatment of vitamin D deficiency in a subject. In preferredembodiments, the methods are performed by sequential competitiveimmunoassay. The Centaur™, Vista™, and Immulite™ are assay systems thatcan be used to perform a competitive immunoassay.

In accordance with the methods for monitoring treatment of vitamin Ddeficiency in a subject, the level of total 25-hydroxyvitamin D2 and25-hydroxyvitamin D3 in a biological sample can be detected by enhancedchemiluminescence (ECL), enzyme immunoassay (EIA), immunohistochemistry(IHC), western blot analysis, radioimmunoassay (RIA),immunofluorescence, equilibrium dialysis, immunodifferentiation, orenzyme-linked immunosorbant assay (ELISA).

In preferred embodiments of the methods for monitoring treatment ofvitamin D deficiency in a subject, the antibody or antigen-bindingfragment used in accordance with the methods is not cross-reactive withvitamin D2 and/or vitamin D3. In preferred embodiments, the antibody orantigen-binding fragment is an antibody or antigen-binding fragment asdescribed herein. For example, the antibody or antigen-binding fragmentcomprises a Lc CDR1 of SEQ ID NO: 26, a Lc CDR2 of SEQ ID NO: 27, and aLc CDR3 of SEQ ID NO: 28, a Hc CDR1 of SEQ ID NO: 10, a Hc CDR2 of SEQID NO: 11 and a Hc CDR3 of SEQ ID NO: 12. In some embodiments, theantibody or antigen-binding fragment has the property of equimolarrecognition for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. Inpreferred embodiments, the antibody is monoclonal antibody 10H9.

Antibodies and antigen-binding fragments that recognize25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 that can be used in themethods for monitoring treatment of vitamin D deficiency in a subjectcan be labeled, for example, with a detectable label. Exemplary labelsinclude but are not limited to chemiluminescent compounds (e.g., anacridinium ester compound), a phosphorescent compound, a fluorescentcompound, a radiolabel, biotin, or an enzyme. The mentioned exemplarylabels can usually only be detected when excited by methods that includebut are not limited to addition of different chemicals, stimulation bylight or exposure to substrate or other compounds. When using acridiniumester compound, chemiluminescence is triggered by peroxide and acid/baseresulting in a flash that can be read by appropriate instrumentation. Anoptional wash step may be used before initiating detectability of thedetectable label.

The antibody or antigen-binding fragment can be immobilized on a solidphase support.

The antibody or antigen-binding fragment can be conjugated to a carrierprotein. The complex between the antibody or antigen-binding protein andcarrier protein also can be immobilized on a solid phase support.

Solid phase supports for use in the methods described herein includeparamagnetic particles; cross-linked dextran available under thetrademark SEPHADEX (Pharmacia Fine Chemicals, Piscataway, N.J.);agarose; polystyrene beads; polyvinyl chloride, polystyrene,cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such assheets, strips or paddles; or tubes, plates or the wells of a microtiterplate such as those made from polystyrene or polyvinylchloride. Whenusing paramagnetic particles, some source of a magnetic field may beused to retain the particles and molecules bound directly or indirectlyto the particles during an optional wash step. The molecules may bebound covalently, by salt-bridges, hydrogen bonding or another type ofbond.

The biological sample can be blood, blood serum or blood plasma. In someembodiments, the biological sample may be stored under biologicalconditions for up to 24 hours prior to use in the methods describedherein.

In some embodiments of the methods for monitoring treatment of vitamin Ddeficiency in a subject, the biological sample is treated or combinedwith 8-anilino-1-naphthalene sulfonate (ANS) before contacting thebiological sample with an antibody or antigen-binding fragment thatrecognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3Alternatively, the biological sample can be treated or combined with8-anilino-1-naphthalene sulfonate (ANS) concurrently with contacting thebiological sample with an antibody or antigen-binding fragment thatrecognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The ANSmay be in the form of ANS acid or a salt (e.g., ANS sodium salt, ANSpotassium salt, ANS hemimagnesium salt or ANS ammonium salt). The ANSmay be present, for example, in a displacement buffer. Methanol mayoptionally be used with the 8-anilino-1-naphthalene sulfonate (ANS)either before contacting the biological sample with an antibody orantigen-binding fragment or concurrently with contacting the biologicalsample with an antibody or antigen-binding fragment. Methanol may, forexample, be included in the displacement buffer.

In some embodiments of the methods for monitoring treatment of vitamin Ddeficiency in a subject, the biological sample also is treated orcombined with 8-anilino-1-naphthalene sulfonate (ANS) and ethyleneglycol before contacting the biological sample with an antibody thatrecognizes both 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 orantigen-binding fragment. Alternatively, the biological sample can betreated or combined with 8-anilino-1-naphthalene sulfonate (ANS) andethylene glycol concurrently with contacting the biological sample withan antibody that recognizes both 25-hydroxyvitamin D2 and25-hydroxyvitamin D3 or antigen-binding fragment. The ANS may be in theform of ANS acid or a salt (e.g., ANS sodium salt, ANS potassium salt,ANS hemimagnesium salt or ANS ammonium salt). The ANS and ethyleneglycol may be present in a displacement buffer. Methanol may optionallybe used with the 8-anilino-1-naphthalene sulfonate (ANS) and ethyleneglycol either before contacting the biological sample with an antibodyor antigen-binding fragment or concurrently with contacting thebiological sample with an antibody or antigen-binding fragment. Methanolmay, for example, be included in the displacement buffer.

In some embodiments of the methods for monitoring treatment of vitamin Ddeficiency in a subject, a 25-hydroxyvitamin D analog is added to thebiological sample following the contacting step. The 25-hydroxyvitamin Danalog can be labeled. 25-hydroxyvitamin D analog or labeled25-hydroxyvitamin D analog can also be present in a stabilization buffercomprising 8-anilino-1-naphthalene sulfonate (ANS). The ANS may be inthe form of ANS acid or a salt (e.g., ANS sodium salt, ANS potassiumsalt, ANS hemimagnesium salt or ANS ammonium salt).

The vitamin D analogs described herein may be based on the use of avitamin D 22 carbon derivative (vitamin D-C22), which includes a C22carboxy group when unconjugated, as depicted in FIG. 1( a) (Hollis etal., Clin. Chem. 39(3):529-33 (1993)). In some embodiments, the vitaminD analog may be vitamin D-C22. In some embodiments, the vitamin D analogmay be conjugated to a carrier protein.

In some embodiments of the methods for monitoring treatment of vitamin Ddeficiency in a subject, the described vitamin D analogs may be affixeddirectly to the protein carrier. For example, vitamin D-C22 may bedirectly conjugated to bovine serum albumin (BSA). The number of vitaminD analogs that may be conjugated to a given protein carrier will varybased on the carrier used. For example, BSA will accommodate the linkageof a relatively modest number of proteins, perhaps about 10 to about 25;alternatively, a carrier such as keyhole limpet hemocyanin (KLH) mayaccommodate about 200 to about 300 antigenic molecules. Those skilled inthe art will understand that a wide variety of carrier proteins may beused for the purposes described herein. Some suitable carriers include,KLH, PEGylated KLH, Concholepas concholepas hemocyanin (CCH), cationizedBSA, and ovalbumin to name only a few.

Carrier protein conjugation to the vitamin D analog may occur throughthe use of a chemical linker. The conjugation of the vitamin D analog orvitamin D derivative to the protein may be achieved by reaction betweenamino groups of the protein and a reactive N-hydroxysuccinimide ester(NHS ester) group of the vitamin D analog or vitamin D derivative.

In some embodiments of the methods for monitoring treatment of vitamin Ddeficiency in a subject, vitamin D levels can be detected in 20 minutesor less starting from the point at which the biological sample iscombined with the displacement buffer.

Vitamin D deficiency in the subject can be indicative of or associatedwith a disease. Diseases associated with vitamin D deficiency caninclude: rickets, osteomalacia high blood pressure, osteoporosis,autoimmune disease, cardiovascular disease, schizophrenia, depression,nervous system disease, diabetes, infectious disease, asthma, allergy orcancer.

Also provided herein are methods for stabilizing 25-hydroxyvitamin Danalog by contacting the 25-hydroxyvitamin D analog with8-anilino-1-naphthalene sulfonate (ANS). The ANS may be in the form ofANS acid or a salt (e.g., ANS sodium salt, ANS potassium salt, ANShemimagnesium salt or ANS ammonium salt). 25-hydroxyvitamin D analogstabilized with 8-anilino-1-naphthalene sulfonate (ANS) can be storedfor greater than 2 months outside an assay system. 25-hydroxyvitamin Danalog stabilized with 8-anilino-1-naphthalene sulfonate (ANS) can bestored for greater than 7 days inside an assay system.

Also provided herein are methods for detecting vitamin D deficiency in asubject. In preferred embodiments, the subject is human. Biologicalsamples for use in the methods can be blood, blood serum or blood plasmaderived from the subject. The methods for detecting vitamin D deficiencyin a subject involve determining the level of total 25-hydroxyvitamin Din a biological sample derived from the subject by combining thebiological sample and a displacement buffer. The biological sample maybe added to the displacement buffer or vice versa to form an assaymixture. The displacement buffer displaces vitamin D from vitamin Dbinding protein. In preferred embodiments, the displacement buffercontains 8-anilino-1-naphthalene sulfonate (ANS). The ANS may be in theform of ANS acid or a salt (e.g., ANS sodium salt, ANS potassium salt,ANS hemimagnesium salt or ANS ammonium salt). The displacement buffermay further contain ethylene glycol. In some embodiments, thedisplacement buffer contains ANS and methanol. In some preferredembodiments, the displacement buffer contains ANS, ethylene glycol, andmethanol.

Next, an antibody or antigen-binding fragment that preferentially binds25-hydroxyvitamin D2, 25-hydroxyvitamin D3, or vitamin D-C22-basedimmunogen conjugated to a first label is combined with the assaymixture. The antibody or antigen-binding fragment may be added to theassay mixture or vice versa and becomes a component thereof. Theantibody that preferentially binds 25-hydroxyvitamin D2, and25-hydroxyvitamin D3, or vitamin D-C22-based immunogen, orantigen-binding fragment thereof, is preferably an antibody orantigen-binding fragment as described above. In preferred embodiments,the antibody or antigen-binding fragment comprises a Lc CDR1 of SEQ IDNO: 26, a Lc CDR2 of SEQ ID NO: 27, and a Lc CDR3 of SEQ ID NO: 28, a HcCDR1 of SEQ ID NO: 10, a Hc CDR2 of SEQ ID NO: 11 and a Hc CDR3 of SEQID NO: 12. In preferred embodiments, the antibody is monoclonal antibody10H9. The antibody or antigen-binding fragment that preferentially binds25-hydroxyvitamin D2, 25-hydroxyvitamin D3, or vitamin D-C22-basedimmunogen can be immobilized on a solid phase support. The antibody orantigen-binding fragment that preferentially binds 25-hydroxyvitamin D2,25-hydroxyvitamin D3, or vitamin D-C22-based immunogen can be conjugatedto a carrier protein. The complex of the antibody or antigen-bindingfragment that preferentially binds 25-hydroxyvitamin D2,25-hydroxyvitamin D3, or vitamin D-C22-based immunogen and the carrierprotein can also be immobilized on a solid phase support.

The first label is preferably a detectable label. The first label can bea chemiluminescent compound (e.g., an acridinium ester compound), aphosphorescent compound, a fluorescent compound, a radiolabel, biotin,or an enzyme. The mentioned exemplary labels can usually only bedetected when excited by methods that include but are not limited toaddition of different chemicals, stimulation by light or exposure tosubstrate or other compounds. When using acridinium ester compound,chemiluminescence is triggered by peroxide and acid/base resulting in aflash that can be read by appropriate instrumentation. An optional washstep may be used before initiating detectability of the detectablelabel.

Next a 25-hydroxyvitamin D analog having a second label is combined withthe assay mixture. The 25-hydroxyvitamin D analog may be added to theassay mixture or vice versa and becomes a component thereof. The secondlabel can be fluorescein for binding to anti-fluorescein antibody,biotin for binding to avidin, streptavidin or anti-biotin antibody,digoxigenin for binding to anti-digoxigenin antibody or other hapten andbinding partner. The 25-hydroxyvitamin D analog can be present in astabilization buffer comprising 8-anilino-1-naphthalene sulfonate (ANS).The ANS may be in the form of ANS acid or a salt (e.g., ANS sodium salt,ANS potassium salt, ANS hemimagnesium salt or ANS ammonium salt). Insome embodiments, the 25-hydroxyvitamin D analog is conjugated to acarrier protein. The carrier protein can be bovine serum albumin,ovalbumin, immunoglobulin, or bovine gamma globulin IgG.

A solid phase support conjugated to an antibody that recognizes thesecond label also is combined with the assay mixture. The solid phasesupport conjugated to the antibody that recognizes the second label maybe added to the assay mixture or vice versa and becomes a component ofthe assay mixture. The antibody that is conjugated to the solid phasesupport can be an antibody that binds to fluorescein.

Solid phase supports for use in the methods described herein includeparamagnetic particles; cross-linked dextran available under thetrademark SEPHADEX (Pharmacia Fine Chemicals, Piscataway, N.J.);agarose; polystyrene particles or beads; polyvinyl chloride,polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-basedwebs such as sheets, strips or paddles; or tubes, plates or the wells ofa microtiter plate such as those made from polystyrene orpolyvinylchloride. When using paramagnetic particles, some source of amagnetic field may be used to retain the particles and molecules bounddirectly or indirectly to the particles during an optional wash step.The molecules may be bound covalently, by salt-bridges, hydrogen bondingor another type of bond.

The level of total 25-hydroxyvitamin D in the biological sample isdetermined by measuring the signal emitted by the first label, wherein areduced level of total 25-hydroxyvitamin D in the biological samplerelative to the level in a normal control or a threshold level of 30ng/mL is indicative of a vitamin D deficiency in the subject.

The immunoreagents of any diagnostic system described herein can beprovided in solution, as a liquid dispersion or as a substantially drypowder, e.g., in lyophilized form.

In some embodiments, vitamin D deficiency can be detected in 20 minutesor less starting from the point at which the biological sample iscombined with the displacement buffer.

The vitamin D deficiency in the subject can be indicative of a disease.The disease can include: rickets, osteomalacia, high blood pressure,osteoporosis, autoimmune disease, cardiovascular disease, schizophrenia,depression, nervous system disease, diabetes, infectious disease,asthma, allergy or cancer.

Kits

Kits can comprise an antibody or antigen-binding fragment as describedherein and instructions, for example, for collecting a biological samplefrom a subject and/or for using the antibody or antigen-binding fragmentto determine the amount of total vitamin D in a biological sample. Inpreferred embodiments, the antibody or antigen-binding fragmentcomprises a detectable label as described herein. The kit can alsocomprise a vitamin D analog linked to a solid support. In someembodiments, the kit may comprise a standard curve or data set showing acorrelation of the quantity or level of vitamin D with normal and/ordeficient vitamin D levels.

The following examples are provided to describe the embodimentsdescribed herein with greater detail. They are intended to illustrate,not to limit, the embodiments.

EXAMPLES Example I Synthesis of Vitamin D-C22 Based Molecules andCompounds

To produce vitamin D-C22 based antigens a manipulable form of themolecule was necessary. To accomplish this, efforts were undertaken toproduce vitamin D-C22 acid via a synthesis scheme based on that ofHollis and Napoli (Clin. Chem, 31:1815-1819 (1985)). Briefly, 23,24-Bisnor-5-cholenic acid-3β-OL acetate (2.50 g) was reacted withmethanol (0.312 mL), dicyclohexylcarbodiimide (1.59 g) andN,N-dimethylaminopyridine (160 mg) in dichloromethane (25 mL) for 3hours to give 23,24-bisnor-5-cholenic acid-3β-OL acetate, methyl ester(1.808 g). The methyl ester (1.808 g) was brominated withN-bromosuccinimide (1.05 g)/azoisobutyronitrile (51.7 mg) in hexane (200mL) under reflux for 30 minutes, followed by dehydrobromination withtetrabutylammonium fluoride (1 M in THF, 23.8 mL) in THF (112 mL) atroom temperature for 2 hours to give 23,24-Bisnor-5,7-choledienicacid-3β-OL acetate methyl ester (1.21 g). 23,24-Bisnor-5,7-choledienicacid-3β-OL acetate, methyl ester (1.21 g) was reacted with potassiumhydroxide (0.50 g) in methanol (18 mL) and ethyl ether (22 mL) at roomtemperature for 2.5 hours to produce 23,24-bisnor-5,7-choledienicacid-3β-OL methyl ester (0.962 g). 23,24-Bisnor-5,7-choledienicacid-3β-OL, methyl ester (0.960 g) was irradiated under 450 w mercurylamp with a Vycor filter in ether (1100 mL) at −10 to 0° C. for 3minutes and 30 seconds twice, then separated by column chromatography onsilica gel to give pre-vitamin D-C22 methyl ester which was refluxed inethanol (100 mL) for 3 hours to produce vitamin D-C22 methyl ester(0.389 g). Vitamin D-C22 methyl ester (249 mg) was reacted withpotassium hydroxide (6.25 g) in methanol (30 mL) at 60° C. for 5 hoursto give vitamin D-C22 acid (165 mg). (FIG. 2.)

To form conjugated compounds with vitamin D-C22 an NHS precursor wasneeded. To achieve this vitamin D-C22 acid (165 mg) was reacted withdicyclohexycarbodiimide (116 mg) and N-hydroxysuccinimide (64 mg) in1,4-dioxane, then reacted with 1,4-diaminobutane (480 uL) at roomtemperature for 2 hours to give vitamin D-DAB (141 mg). Theprotein-reactive reagents, vitamin D-DAB-Suberoyl-NHS and vitaminD-DAB-PEG5-NHS, were prepared from vitamin D-DAB by reaction with excessdisuccinimidyl suberate or PEGS-Di-NHS. The NHS esters were purified bypreparative reversed-phase HPLC through a C18 column. Vitamin D-DAB (30mg) was reacted with excess of disuccinimidyl suberate (DSS, 133 mg) inDMF (1.2 mL) and triethylamine (15 uL) for 3.5 hours. The product (24.5mg) was purified by preparative HPLC through a Synergi Hydro-RP columnto produce vitamin D-DAB-Suberoyl-NHS. Vitamin D-DAB (41 mg) was reactedwith excess of Bis-PEG5-NHS(282 mg) in DMF (2.0 mL) and triethylamine(20 uL) for 3.5 hours. The product (33.7 mg) was purified by preparativeHPLC through a Synergi Hydro-RP column to produce vitaminD-DAB-PEG5-NHS. (FIG. 3.) Protein conjugates were prepared by reactionbetween the NHS ester and the lysine amino groups of the proteins asrepresented in FIG. 4. Vit D-DAB-Suberoyl-BSA was prepared by thereaction of Vit D-DAB-Suberoyl-NHS (5 mg) with BSA (10 mg) in a mixtureof 0.1 M phosphate buffer, ph 7.5 (1 mL) and DMF (0.4 mL) at roomtemperature for 2 h and purified by centrifiltration using PBS, pH 7.2for buffer exchange. MALDI-TOF mass spectrometry showed loading of 14Vitamin D labels per BSA.

Vitamin D-DAB-PEGS-BSA-Fluorescein conjugates (FIG. 5) were prepared viaa two-step conjugation by reaction between the free thiol of BSA withfluorescein-5-maleimide followed by reaction of the lysine amino groupswith vitamin D-DAB-PEG5-NHS. The conjugates were isolated by gelfiltration using a Sephadex G25 column. It was found that the conjugateprepared using a 10:1 fluorescein-5-maleimide to BSA molar ratiofollowed by conjugation at 20:1 vitamin D-DAB-PEG5-NHS to BSA molarratio produced the best Centaur® assay curves.

Vitamin D-fluorescein conjugates were prepared and used as magneticparticle coating antigen for the Centaur® assay. Good immunoassayresults were obtained using a small-molecule derivative, vitaminD-DAB-PEG5-aminopentyl-thioureidyl fluorescein, MW 1208 (structurebelow), as a magnetic particle coating antigen.

Example II Production of Antibodies Reactive to Vitamin D-C22 AntigenicCompounds

Experiments were conducted to produce antibodies that couldpreferentially bind to molecules having structural aspects of thevitamin D-C22 antigenic molecule. These experiments were conducted inaccordance with the method of Galfre et al. (Nature, 266:550 (1977)), asmodified by Oi and Herzenberg (Selected Methods in Cellular Immunology(1980)). Initially, BALB/c mice were immunized with vitamin D-C22 BSAemulsified in Freund's Complete adjuvant followed by a secondaryimmunization using Freund's Incomplete adjuvant. Sera of immunized micewere collected two weeks after the secondary immunization.

Collected sera were tested for antigen reactivity by ELISA as follows:microtiter plates coated with vitamin D-C22 BSA were incubated withmouse antisera, diluted in dilution buffer, for one hour. Plates werewashed and a secondary antibody (goat-anti-mouse IgG) conjugated tohorseradish peroxidase (HRPO) in dilution buffer was added and incubatedfor 30 minutes. Plates were washed and the colorimetric substrate3,3′,5,5′-tetramethylbenzidine (TMB) was added. The generated color wasstopped using 1N sulfuric acid and the optical density was measured at450 nm. Sera from all five mice tested showed substantial reactivityrelative to normal mouse serum (NMS) (Table 2).

TABLE 2 Vitamin D-C22 BSA Detection by Sera from Immunized Mice COAT:Vit D C22 Lc-BSA (JL808) @ 500 ng/mL MOUSE # NMS 0 1 2 3 4 Vit D 1:12800 0.055 2.449 2.406 1.731 2.325 3.042 C22  1:6400 0.056 3.1773.133 2.533 3.057 3.473 Initial  1:3200 0.059 3.534 3.508 3.171 3.4543.608 Bleed  1:1600 0.062 3.578 3.605 3.532 3.638 3.687 May 27, 1:8000.069 3.634 3.731 3.603 3.708 3.696 2008 1:400 0.083 3.602 3.640 3.6333.633 3.441 1:200 0.110 3.750 3.693 3.649 3.663 3.634 1:100 0.174 3.7473.687 3.625 3.700 3.652

Example III Production of Monoclonal Antibodies Reactive to VitaminD-C22 Antigenic Compounds

Mice exhibiting a positive immune response to vitamin D-C22 antigenswere selected for monoclonal antibody development. Briefly, spleen cellsharvested from selected mice were fused with mouse Sp2/0 myeloma cells.Resulting hybridomas that produced antibodies reactive to25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were selected and clonedat least twice by the limiting dilution procedure to obtain monoclonalantibody-producing cell lines. One monoclonal cell line that wasidentified and tested further was the 10H9 hybridoma.

Following isolation of hybridoma clones, studies were conducted todetermine the relative ability of the antibodies to bind to both25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. To assess this, antibodydisplacement assays were conducted to determine the degree to whichbinding between vitamin D-C22 BSA and a reactive antibody raised againstthe antigen (10H9) could be disrupted by the presence of25-hydroxyvitamin D2 or 25-hydroxyvitamin D3. Cell culture supernatantfrom the 10H9 hybridoma cell lines producing antibodies reactive tovitamin D-C22 BSA were coincubated in the presence or absence of25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 on microtiter plates coatedwith vitamin D-C22 KLH for 1 hour. After washing the plates, goatanti-mouse IgG-HRPO was added and incubated for 30 minutes. Plates werewashed and then incubated with tetramethyl benzidine (TMB). Thegenerated color was stopped using 1N sulfuric acid and the opticaldensity was measured at 450 nm. As set forth in Table 3 (and as depictedin FIG. 6), antibody binding to vitamin D-C22 BSA was disrupted in aconcentration-dependent manner by coincubation with either25-hydroxyvitamin D2 or 25-hydroxyvitamin D3. Both 25-hydroxyvitamin D2and 25-hydroxyvitamin D3 showed substantially similar binding disruptionprofiles which is characteristic of equimolar affinity of the antibodytested.

TABLE 3 Vitamin D-C22-Reactive Antibody Displacement by25-hydroxyvitamin D2 or 25-hydroxyvitamin D3. Summary of Vit D C22-BSAsuper displacement assay Coat: Vit D C22-Diaminobutane-Suberoyl-KLH (LotJL814) @ 50 ng/mL 25 OH-D2 25 OH-D3 Ag @ (Final conc.) 0 0.4 μg/mL 0.4μg/mL Vit D C22-BSA 1:4  1.997 0.323 0.384 10H9 @ (Final 1:16 1.2440.154 0.176 dilution) 1:64 0.672 0.097 0.105  1:256 0.313 0.086 0.090Tracer: GAM-IgG-HRP (Fc) @ 1:20K

Next, displacement experiments were conducted using purified antibody,rather than cell supernatant. In this experiment, the antibody ofinterest was coated directly on the microtiter plate via a two-hourincubation. Coated plates were washed and then coincubated with either25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 in the presence of alkalinephosphatase-conjugated vitamin D-C22 BSA. After 30 minutes the plateswere washed and color was developed by adding the substratep-nitrophenyl phosphate (PNPP). Optical density colored solution wasmeasured at 405 nm. Again, 10H9 binding to vitamin D-C22 BSA wasdisrupted in a concentration-dependent manner by coincubation witheither 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 (Table 4). Both25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 showed substantiallysimilar binding disruption profiles which is characteristic of equimolaraffinity of the antibody tested (FIG. 7).

TABLE 4 Vitamin D-C22-Reactive Antibody Displacement by25-hydroxyvitamin D2 or 25-hydroxyvitamin D3. Coat 50 ul MAb @ 1 ug/mL10H9 25 uL of 0 1.277 25OH-VIT D2 5 1.189 (Fluka 1402454- 10 1.00713808245- 25 0.771 090319MR) 100 0.106 @ indicated final 200 0.080concentrations in 400 0.069 ng/ml 1000 0.063 25 uL of 0 1.213 25OH-VITD3 5 1.083 (Fluka 1412212- 10 0.800 24708001- 25 0.519 090323GK) @ 1000.115 indicated final 200 0.089 concentrations in 400 0.077 ng/ml 10000.069

Example IV Vitamin D Assay

Biological sample is added to a reaction cuvette followed bydisplacement buffer and allowed to react for 4.5 minutes. Monoclonalantibody conjugated to acridinium ester is added and allowed to reactfor 5.5 minutes to bind 25-hydroxyvitamin D in the sample. A25-hydroxyvitamin D analog conjugated to bovine serum albumin andfluorescein is added along with antifluorescein-coated paramagneticparticles and allowed to react for 3.75 minutes. The reaction cuvette iswashed, and acid and base reagents are added to initiate thechemiluminescent reaction. The time-to-result is 18 minutes. An inverserelationship exists between the amount of 25-hydroxyvitamin D in thepatient sample and the amount of relative light units (RLUs) detected bythe system.

Assay:

The ADVIA Centaur Vitamin D Total assay is a one-pass, 18-minuteantibody competitive immunoassay that uses an antifluorescein-labeledmonoclonal antibody covalently bound to paramagnetic particles (PMPs),one monoclonal antibody labeled with acridinium ester (AE), and avitamin D analog labeled with fluorescein (FIG. 8). The Vitamin D Totalassay requires 20 L. of sample volume for a single determination. Thetime-to-first-result is 18 minutes and the throughput is 240 tests/hour.

Briefly, the first step of the sequential step hapten/antibodycompetitive immunoassay using chemiluminescent technology begins withthe analyzer dispensing 20 μL of biological sample into a cuvettefollowed by addition of 200 μl of displacement buffer (50 mM HEPES, 150mM NaCl, 0.09% sodium azide, pH=7.5) containing8-anilino-1-naphthalenesulfonic acid ammonium salt (Sigma-aldrich, St.Louis, Mo.) and ethylene glycol (Sigma-aldrich, St. Louis, Mo.) andincubation for 4.5 minutes at 37° C. Lite Reagent (50 μL) containing ananti 25-hydroxy Vitamin D monoclonal antibody labeled with acridiniumester (monoclonal antibody 10H9) is added to the mixture and incubatedfor 5.5 min at 37° C. C22-PEG-BSA-fluorescein conjugate (50 μL) andanti-fluorescein monoclonal antibody coated paramagnetic microparticles(100 μL) are added to the mixture and incubated for 3.75 minutes at 37°C. The fourth step is separation of Solid Phase complex with boundC22-PEG-BSA-fluorescein conjugate and anti 25-hydroxy vitamin Dmonoclonal antibody labeled with acridinium ester, followed by washingthree times to remove any free Lite Reagent. Last is the sequentialdispensing of 300 μL each of acid reagent then base reagent to initiatethe chemiluminescent reaction. The total incubation time from sample toresult is 18 minutes. There is an indirect relationship between theamount of 25-hydroxy vitamin D present in the biological sample and theamount of chemiluminescence quantified as relative light units (RLUs).In the Centaur-based assay, there is an inverse relationship betweenRLUs emitted by the acridinium ester and the amount of vitamin D becausethe 25-hydroxyvitamin D released from the carrier protein in the plasmacompetes with the Vitamin D-BSA-Fluorescein for binding with a limitedamount of the acridinium-labeled 10H9 monoclonal antibody. Higher levelsof 25(OH)D in the patient sample would lower the amount of theacridinium-MAb-VitD-BSA-Fluorescein complex on the magnetic particlesresulting in lower RLUs.

Precision:

The precision study was based on the CLSI protocol EP5-A2: two runs perday for 10 days on a single ADVIA Centaur system. Assay precision wasdetermined using samples with Vitamin D Total ranging from 4 to 120ng/mL.

Analytical Sensitivity:

Analytical sensitivity is defined as the concentration that correspondsto the mean signal plus 2 SD obtained from the lowest standard,expressed in relative light units (RLUs). The analytical sensitivitystudy was performed following CLSI protocol EP5-A2. Analyticalsensitivity was determined using 60 replicates of the lowest standard.

Limit of Blank, Limit of Detection, and Functional Sensitivity:

The limit of blank is defined as the concentration of analyte thatcorresponds to the 95th percentile of the distribution of a humannegative basepool. The vitamin D total low standard was assayed 20 timesusing two lots of reagents on three systems (n=120). The limit ofdetection (LoD) is determined according to CLSI protocol EP 17-A. Limitof detection is defined as the lowest concentration of vitamin D thatcan be detected with 95% probability. The LoD was determined by usinglow-level vitamin D samples that were assayed 20 times using two lots ofreagents on three systems (n=120). Functional sensitivity was determinedusing a single instrument over 10 days. Two runs were performed per dayin duplicate for a total of 60 replicates. The ADVIA Centaur Vitamin DTotal sensitivity panel members concentrations ranged from 3.0 to 20.0ng/mL. Concentrations were calculated using within-lot, day zero,two-point calibration curves.

Interference Studies:

Interference from endogenous and nonendongenous substances wereevaluated following the guidelines in NCCLS EP-7A. Each sample wasspiked with an interferent and compared to a matched unspiked control.

Cross-Reactivity:

Five vitamin D derivatives were analyzed using the ADVIA Centaur VitaminD Total assay. The vitamin D derivatives were spiked into a samplecontaining 27 ng/mL of vitamin D total. Three replicates of the spikedsamples were assayed and the vitamin D total concentration determined

Tube Type Study:

Correlation of EDTA and serum separator tubes (SST) tubes was analyzedusing the ADVIA Centaur Vitamin D Total assay. Serum red top, SST, andEDTA tubes were collected from 119 donors and assayed using the CentaurVitamin D Total assay. Three replicates of each sample were evaluated.Linear regression correlation between serum and SST and serum vs EDTAwas determined.

Method Correlation:

The ADVIA Centaur Vitamin D Total assay was compared to a commerciallyavailable, FDA-cleared, Vitamin D Total immunoassay using 199 patientspecimens, a single replicate for each method. Specimen concentrationranged from 5 to 150 ng/mL. In addition, 23 patient samples were assayedby LC-MS/MS and the ADVIA Centaur Vitamin D Total Assay. Specimens inthis second population ranged from 11 to 82 ng/mL.

Results

The data obtained with the ADVIA Centaur Vitamin D Total assaydemonstrated equimolar detection of 25(OH)D₂ and 25(OH)D₂ and showedtraceability to LC-MS/MS. Cross-reactivity to 25(C1H)D₂ was determinedto be 105% at 50 ng/mL. The assay demonstrated a limit of detection(LoD) of less than 3.0 ng/mL, a functional sensitivity (20% dose totalCV) of less than 4 ng/mL, and an upper limit of 250 ng/mL. Total assayCVs were 6.4%, 7.1%, 4.2%, and 3.7% for samples at 22.1, 52.3, 121, and153 ng/mL, respectively. Linearity up to 240 ng/mL was demonstrated. Acorrelation study against LC-MS/MS was performed with 150 serum samples,yielding a slope of 0.96, intercept of 1.0, and regression coefficientof 0.97.

Precision:

The precision profile of the ADVIA Centaur Vitamin D Total assaydemonstrates a total CV between 8.8% at 7.65 ng/mL to 2.0% at 123.36ng/mL 25(OH) total vitamin D. Precision analysis is shown in Table 5.

TABLE 5 Precision analysis of ADVIA Centaur Vitamin D Total Assay MeanWithin-Run Within-Run Total SD Total Sample (ng/mL) SD (ng/mL) CV (%)(ng/mL) CV (%) 1 7.65 0.65 8.5 0.67 8.8 2 10.65 0.85 8.0 1.07 10.1 313.11 0.84 6.4 0.91 6.9 4 15.87 1.02 6.4 1.18 7.4 5 18.40 1.31 7.1 1.447.8 6 22.63 1.79 7.9 1.79 7.9 7 59.75 1.76 3.0 1.92 3.2 8 99.63 1.95 2.02.07 2.1 9 112.74 1.98 1.8 3.07 2.7 10 115.71 1.98 1.7 2.55 2.2 11123.36 2.29 1.9 2.51 2.0

Analytical Sensitivity:

The analytical sensitivity of the ADVIA Centaur Vitamin D Total assaywas 2.4 ng/mL. Analytical sensitivity is shown in Table 6.

TABLE 6 Analytical sensitivity of ADVIA Centaur Vitamin D Total AssaySample Replicates Mean RLU + 2 SD Dose (ng/mL) Vitamin D 60 688200 2.4Blank

Limit of Blank, Limit of Detection, and Functional Sensitivity:

The limit of blank of the ADVIA Centaur Vitamin D Total assay was 2.8ng/mL, the limit of detection was 3.8 ng/mL, and the functionalsensitivity was 4 ng/mL (FIG. 9).

Interference Studies:

The ADVIA Centaur Vitamin D Total assay demonstrated ≦10% bias at theconcentrations tested for endogenous interferents. Results of endogenousinterference studies is shown in Table 7.

TABLE 7 Interference Study Results for ADVIA Centaur Vitamin D TotalAssay Vitamin D Vitamin D Total Total Expected Observed Bias InterferentConcentration (ng/mL) (ng/mL) (%) Unconjugated  60 mg/dL 30.88 32.675.77 Bilirubin Conjugated  60 mg/dL 33.82 30.67 — Bilirubin 9.31 Albumin 9 g/dL 22.5 20.4 — 9.33 Hemoglobin 500 mg/dL 29.08 29.70 2.13Triglycerides 500 mg/dL 22.7 23.6 3.96 Uric Acid  20 mg/dL 35.45 33.45 —5.64

Cross-Reactivity:

The ADVIA Centaur Vitamin D Total assay demonstrated very lowcross-reactivity to the nonhydroxylated forms of vitamin D2 and vitaminD3, and to 3-epi-25(OH)D3. Cross-reactivity analysis results are shownin Table 8.

TABLE 8 Cross-reactivity of ADVIA Centaur Vitamin D Total Assay ExpectedObserved (Endogenous) Vitamin Cross- Concentration Vitamin D D Totalreactivity Crossreactant (ng/mL) Total (ng/mL) (ng/mL) (%) 25-(OH)-Vit27 0 27 100 D3 25-(OH)-Vit 30 27 58 102 D2 Vitamin D2 100 27 28 0.04Vitamin D3 100 27 28 0.04 3-epi- 100 27 27 0.0 25(OH)D3

Tube Type Study:

A sample tube type correlation was performed with 119 donor specimenscollected in serum red top, SST, and EDTA tube types. Regressionanalysis between serum red top and SST demonstrates a correlationcoefficient (R) of 0.999, a slope of 1.01, and an intercept of −0.14(FIG. 10). Regression analysis between serum red top and EDTAdemonstrates a correlation coefficient (R) of 0.997, a slope of 1.00,and an intercept of 0.41 (FIG. 11).

Method Comparison:

A sample correlation was performed with 199 specimens comparing theADVIA Centaur Vitamin D Total assay to a commercially available,FDA-cleared, Vitamin D Total assay. Regression analysis demonstrated acorrelation coefficient (R) of 0.993, a slope of 1.00, and an interceptof 1.61 (FIG. 12). In, addition 23 specimens were assayed comparing theADVIA Centaur Vitamin D Total assay to a commercially available VitaminD Total LC-MS/MS assay. Regression analysis demonstrated a correlationcoefficient (R) of 0.98, a slope of 1.03, and an intercept of −2.3 (FIG.13).

SEQ ID NO. Sequence  1 TTTTGGGGCCAAGGCACCACTCTCACAGTCTCCTCG  2TACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAAC  3 TTTACTATCTATAATCAGAAG  4ATAAGAGCGCATTACGACGGGAGAGTT  5GTGCAGCTGCTCGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGA C  6CTTGAGTGGATTGGACTTATTAATCCTTACAATGGT  7TTCAAGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCCTCAGTCTGACATCTGAAGACTCTGCAGT CTATTACTTT  8GTGCAGCTGCTCGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAATGGTTTTACTATCTATAATCAGAAGTTCAAGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCCTCAGTCTGACATCTGAAGACTCTGCAGTCTATTACTTTATAAGAGCGCATTACGACGGGAGAGTTTTTTGGGGCCAAGGCACCACTCTCACAGTCTCCTCG  9 FWGQGTTLTVSS 10 YTMNWVKQSHGKN11 FTIYNQK 12 IRAHYDGRV 13 VQLLESGPELVKPGASMKISCKASGYSFTD 14LEWIGLINPYNG 15 FKGKATLTVDKSSSTAYMELLSLTSEDSAVYYF 16VQLLESGPELVKPGASMKISCKASGYSFTDYTMNWVKQSHGKNLEWIGLINPYNGFTIYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAV YYFIRAHYDGRVFWGQGTTLTVSS17 ACGTTCGGAGGGGGGACCAAGCTAGAAATAAAACGG 18CAGAGCCTTGTACACAGTAATGGAAACACCTATTTACAT 19 CTGATCTACCAAGTTTCCAAC 20TGCTCTCAAATTACACATTTTCCTCCC 21TGTGAACTAGTGATGACCCAGTCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCGTCTCTTGCAGATCTAGT 22CGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTC 23CGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCACCAGAGTGGAGGCTGAGGATC TGGGAGTTTATTTC 24TGTGAACTAGTGATGACCCAGTCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCGTCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATCGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACCAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCACCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAATTACACATTTTCCTCCCACGTTCGGAGGGGGGACCAAGCTAGAAATAAAACGG 25 TFGGGTKLEIKR 26 QSLVHSNGNTYLH 27LIYQVSN 28 CSQITHFPP 29 CELVMTQSPLSLPVSLGDQASVSCRSS 30 RYLQKPGQSPKL 31RFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYF 32CELVMTQSPLSLPVSLGDQASVSCRSSQSLVHSNGNTYLHRYLQKPGQSPKLLIYQVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYF CSQITHFPPTFGGGTKLEIKR

What is claimed:
 1. An isolated antibody, or an antigen-binding fragmentthereof, comprising a heavy chain CDR1 having the amino acid sequence ofSEQ ID NO: 10, a heavy chain CDR2 having the amino acid sequence of SEQID NO: 11, a heavy chain CDR3 having the amino acid sequence of SEQ IDNO: 12, a light chain CDR1 having the amino acid sequence of SEQ ID NO:26, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 27,and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 28,wherein the antibody binds 25-hydroxyvitamin D2 and 25-hydroxyvitaminD3.
 2. The antibody or antigen-binding fragment thereof of claim 1,wherein the heavy chain variable domain comprises the amino acidsequence of SEQ ID NO:
 16. 3. The antibody or antigen-binding fragmentthereof of claim 1, wherein the light chain variable domain comprisesthe amino acid sequence of SEQ ID NO:
 32. 4. The antibody of claim 1,wherein the antibody is a murine antibody.
 5. The antibody of claim 1,wherein the antibody is recombinant.
 6. The antibody of claim, whereinbinding to 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 is withsubstantially similar affinity.
 7. The antibody of claim 1, wherein theantibody is produced by injecting a mammal with an immunogen comprisingvitamin D-C22.
 8. The antibody of claim 7, wherein the immunogen furthercomprises a carrier protein and a linker that comprises at least 6 atomsin a linear chain.
 9. The antibody of claim 7, wherein the immunogen isrepresented by the formula:

wherein x and y are, independently, 1-12, and at least onelinker-conjugated vitamin D-C22 molecule is bound to the carrierprotein.
 10. The antibody of claim 7, wherein the immunogen isrepresented by the formula:

wherein at least one linker-conjugated vitamin D-C22 molecule is boundto the bovine serum albumin (BSA) carrier protein.
 11. The antibody ofclaim 7, wherein the immunogen is represented by the formula:

wherein at least one linker-conjugated vitamin D-C22 molecule is boundto the keyhole limpet hemocyanin (KLH) carrier protein.
 12. An isolatedcell expressing the antibody of claim
 1. 13. The cell of claim 12,wherein the cell is derived from an immunized mouse.
 14. The cell ofclaim 13, wherein the cell is a hybridoma.
 15. The cell of claim 12,wherein the cell is recombinantly produced.
 16. An isolatedpolynucleotide encoding the antibody of claim
 1. 17. The isolatedpolynucleotide of claim 16, wherein the polynucleotide is recombinant.18. The isolated polynucleotide of claim 16, wherein the polynucleotideis a cDNA.
 19. A method for detecting vitamin D deficiency in a subject,said method comprising: contacting a biological sample derived from saidsubject with the antibody of claim 1 or antigen binding fragmentthereof; determining the level of total 25-hydroxyvitamin D in thebiological sample; wherein a decrease between the level in thebiological sample and the level in a normal control or a threshold levelof 30 ng/mL is indicative of a vitamin D deficiency in said subject. 20.A method for treating a subject suspected of having a vitamin Ddeficiency, said method comprising: contacting a biological samplederived from said subject with the antibody of claim 1 or antigenbinding fragment thereof; determining the level of total25-hydroxyvitamin D in the biological sample; and if a decrease betweenthe level in the biological sample relative to the level in a normalcontrol or a threshold level of 30 ng/mL is determined, administering tosaid subject a treatment for vitamin D deficiency.
 21. A method formonitoring progression of vitamin D deficiency in a subject in needthereof, said method comprising: contacting a first biological samplederived from said subject with the antibody of claim 1 or antigenbinding fragment thereof; determining the level of total25-hydroxyvitamin D in the first biological sample derived from saidsubject at a first time; contacting a second biological sample derivedfrom said subject with the antibody of claim 1 or antigen bindingfragment thereof; and, determining the level of total 25-hydroxyvitaminD in the second biological sample derived from said subject at a secondtime later than said first time; wherein a decrease between the level inthe first biological sample and the level in the second biologicalsample is indicative of the progression of a vitamin D deficiency insaid subject, wherein little or no change between the level in the firstbiological sample and the level in the second biological sample isindicative of stabilization of a vitamin D deficiency in the subject,and wherein an increase between the level in the first biological sampleand the level in the second biological sample is indicative ofregression of a vitamin D deficiency in the subject.
 22. A method formonitoring treatment of vitamin D deficiency in a subject in needthereof, said method comprising: contacting a first biological samplederived from said subject with the antibody of claim 1 or antigenbinding fragment thereof; determining the level of total25-hydroxyvitamin D in the first biological sample derived from saidsubject at a first time; contacting a second biological sample derivedfrom said subject with the antibody of claim 1 or antigen bindingfragment thereof; and, determining the level of total 25-hydroxyvitaminD in the second biological sample derived from said subject at a secondtime later than said first time and following treatment of said subjectfor said vitamin D deficiency; wherein an increase in or stabilizationof the level in the second biological sample relative to the level inthe first biological sample is indicative of efficacy of the treatmentof the vitamin D deficiency in said subject, and wherein a decrease inthe level in the second biological sample relative to the level in thefirst biological sample is indicative of inefficacy of the treatment ofthe vitamin D deficiency in said subject.
 23. A method for detectingvitamin D deficiency in a subject by determining the level of total25-hydroxyvitamin D in a biological sample derived from said subject,said method comprising: combining a biological sample and a displacementbuffer in a vessel; adding to said vessel the antibody of claim 1 orantigen-binding fragment thereof conjugated to a first label; adding tosaid vessel a 25-hydroxyvitamin D analog conjugated to a carrier proteinand a second label; adding to said vessel a solid phase supportconjugated to an antibody that recognizes the second label; andmeasuring the level of total 25-hydroxyvitamin D in the biologicalsample; wherein a decrease between the level in the biological sampleand the level in a normal control or a threshold level of 30 ng/mL isindicative of a vitamin D deficiency in said subject.
 24. A hybridomacell that produces the antibody of claim 1, or an antigen-bindingfragment thereof.
 25. A kit comprising the antibody of claim 1, or anantigen-binding fragment thereof.
 26. A diagnostic device comprising theantibody of claim 1, or an antigen-binding fragment thereof.
 27. The kitof claim 25 further comprising an alkaline phosphatase-conjugatedvitamin D-C22-bovine serum albumin (BSA).
 28. The diagnostic device ofclaim 26 further comprising an alkaline phosphatase-conjugated vitaminD-C22-bovine serum albumin (BSA).
 29. The device of claim 26 whereinsaid device performs enhanced chemiluminescence (ECL), enzymeimmunoassay (EIA), immunohistochemistry (IHC), western blot analysis,radioimmunoassay (RIA), immunofluorescence, equilibrium dialysis,immunodifferentiation, or enzyme-linked immunosorbant assay (ELISA).